Proteolytic inactivation of select proteins in bacterial extracts for improved expression

ABSTRACT

The present disclosure provides modified proteins that are capable of being cleaved by the protease OmpT1. The proteins can be modified in an exposed surface motif to incorporate OmpT1 cleavage sites. Also provided are nucleic acids encoding the modified proteins, bacterial cells that express the modified proteins, and cell free synthesis systems containing modified RF1. The disclosure further provides methods for reducing the deleterious activity of a modified protein in a cell free synthesis system by contacting the modified protein with OmpT1. Also provided are methods for reducing RF1 competition at an amber codon in the cell free synthesis system, and methods for expressing a protein in the cell free synthesis system. The modified proteins of the invention can be used to increase the yield of proteins having non-natural amino acids incorporated at an amber codon.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present patent application is a Continuation of U.S. applicationSer. No. 14/434,391, filed Apr. 8, 2015, now U.S. Pat. No. 9,650,621,which is a U.S. National Phase application under 35 U.S.C. 371 ofApplication No. PCT/US13/63804, filed Oct. 8, 2013, which claims benefitof priority to U.S. Provisional Application No. 61/713,245, filed Oct.12, 2012, which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“091200-1043302-005620US_SEQ.txt” created May 4, 2017, and containing60,908 bytes. The material contained in this text file is incorporatedby reference in its entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to cell free synthesis technology, andparticularly to the proteolytic inactivation of select proteins in abacterial extract. A preferred use is to increase the yield ofpolypeptides having a non-native amino acid incorporated at a definedamino acid residue.

BACKGROUND OF THE INVENTION

The use of bacterial cell-free extracts for in vitro protein synthesisoffers several advantages over conventional in vivo protein expressionmethods. Cell-free systems can direct most, if not all, of the metabolicresources of the cell towards the exclusive production of one protein.Moreover, the lack of a cell wall and membrane components in vitro isadvantageous since it allows for control of the synthesis environment.However, the efficiency of cell-free extracts can be decreased bybacterial proteins that inhibit protein synthesis, either directly orindirectly. Thus, inactivation of undesirable proteins that decrease theefficiency of protein synthesis should increase the yield of desirableproteins in cell-free extracts. For example, the inactivation ofproteins that decrease the efficiency of protein synthesis shouldincrease the yield of polypeptides having non-native amino acidsincorporated at a defined amino acid residue. The introduction ofnon-native amino acids (nnAA) into polypeptides is useful for increasingthe biological diversity and function of proteins. One approach forproducing polypeptides having a nnAA incoroporated at a defined aminoacid residue is to use an nnAA, aminoacylated orthogonal CUA containingtRNA for introduction of the nnAA into the nascent polypeptide at anamber (stop) codon during protein translation. However, theincorporation of nnAA at an amber codon can be inhibited by the nativebacterial termination complex, which normally recognizes the stop codonand terminates translation. Release Factor 1 (RF1) is a terminationcomplex protein that facilitates the termination of translation byrecognizing the amber codon in an mRNA sequence. RF1 recognition of theamber stop codon can promote pre-mature truncation products at the siteof non-native amino acid incorporation, and thus decreased proteinyield. Therefore, attenuating the activity of RF1 may increase nnAAincorporation into recombinant proteins.

It has previously been shown that nnAA incorporation can be increased byattenuating RF1 activity in 3 ways: 1) neutralizing antibodyinactivation of RF1, 2) genomic knockout of RF1 (in an RF2 bolsteredstrain), and 3) site specific removal of RF1 using a strain engineeredto express RF1 containing a protein tag for removal by affinitychromatography (Chitin Binding Domain and His Tag). The presentdisclosure describes a novel method for inactivating RF1 by introducingproteolytic cleavage sites into the RF1 amino acid sequence. Thecleavage sites are not accessible to the protease during bacterial cellgrowth, but are cleaved by the protease when the bacterial cells arelysed to produce cell-free extract. Thus, the yield of full lengthpolypeptides having a nnAA incorporated at an amber codon is increasedin bacterial cell extracts expressing modified RF1 variants describedherein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides recombinant target proteins that aremodified to comprise an Outer Membrane Protein T1 (OmpT1) proteasecleavage site. The OmpT1 cleavage site includes a scissile OmpT1 peptidebond. In some embodiments, the target protein is modified to introducethe OmpT1 cleavage site into a surface exposed motif of the targetprotein. Because OmpT1 cleaves substrates between dibasic residues withhigh efficiency, the native surface exposed motif is modified to includetwo adjacent basic amino acids that are positively charged at pH 7.0.The target protein can be an essential protein that is required, forexample, for normal cell growth and/or survival.

In some embodiments, the surface exposed motif has a B factor of atleast 50 Å² when the motif region of the protein is uncomplexed. In someembodiments, surface exposed motif has a total solvent accessiblesurface area of between about 25 Å² and about 225 Å². In someembodiments, the target protein is modified such that the scissile OmpT1peptide bond is located in a position of the protein which exhibits aPhi angle of from 0° to −180° or a Psi angle from 0° to +180° in aRamachadran Plot.

In one aspect, the invention provides a bacterial cell expressing bothOmpT1 and an essential target protein recombinantly modified to includea scissile OmpT1 peptide bond. In some embodiments, the scissile OmpT1peptide bond is located within a surface exposed motif and the nativemotif is modified to include two adjacent basic amino acids that arepositively charged at pH 7.0. In some embodiments, the bacterial cellexpresses an essential target protein recombinantly modified to includea scissile OmpT1 peptide bond where the bond is in a position of theprotein which is located within a surface exposed motif having a Bfactor of at least 50 Å² when the protein is uncomplexed. In someembodiments, the bacterial cell expresses an essential target proteinrecombinantly modified to include a scissile OmpT1 peptide bond wherethe bond is in a position of the protein which is located within asurface exposed motif having a total solvent accessible surface area ofbetween about 25 Å² and about 225 Å². In one embodiment, the bacterialcell expresses an essential target protein recombinantly modified toinclude a scissile OmpT1 peptide bond where the bond is in a position ofthe protein which exhibits a Phi angle of from 0° to −180° or a Psiangle from 0° to +180° in a Ramachadran Plot. In one embodiment, thebacterial cell is an E. coli.

In some embodiments, the essential target protein is selected from RF1,RF2, RNAse, thioredoxin reductase, glutarodoxin reductase, glutathionereductase, amino acid degrading enzymes, polyphosphate kinase, or coldshock proteins.

In a second aspect, the invention provides a method for reducing thedeleterious activity of a modified essential target protein in an invitro cell free synthesis system, the method comprising the steps of:

-   -   i) culturing an OmpT1 positive bacteria expressing the modified        essential target protein said protein modified to include a        scissile OmptT1 peptide bond where the protein is modified to        include two adjacent basic amino acids that are positively        charged at pH 7.0;    -   ii) lysing the bacteria to create a cell free synthesis extract;    -   iii) contacting the essential target protein with OmpT1 in an        amount sufficient to reduce intact protein by 50%;    -   iv) adding a nucleic acid template to the extract where the        template codes for a protein of interest; and,    -   v) allowing the cell free synthesis system to produce the        protein of interest.

In some embodiments of the method, the scissile OmptT1 peptide bond islocated within a surface exposed motif having a B factor of at least 50Å² when the protein is uncomplexed. In some embodiments, the scissileOmpT1 peptide bond is located within a surface exposed motif having atotal solvent accessible surface area of between about 25 Å² and about225 Å². In some embodiments, the scissile OmpT1 peptide bond is locatedin a position of the protein which exhibits a Phi angle of from 0° to−180° or a Psi angle from 0° to +180° in a Ramachadran Plot.

In some embodiments of the method, the oxidative phosphorylation systemof the bacteria remains active after cell lysis and during the synthesisof the protein of interest. In one embodiment, the cell free synthesissystem places a non-native amino acid at an amber codon of the proteinof interest.

In a third aspect, the invention provides a functional Releasing Factor1 protein (RF1) that is cleavable by an Outer Membrane Protein T1(OmpT1), where a scissile OmpT1 peptide bond is located within theswitch loop region corresponding to amino acids 287-304 of wild type RF1(SEQ ID NO:1) and where the switch loop region is modified to includetwo adjacent basic amino acids that are positively charged at pH 7.0. Insome embodiments, the two adjacent basic amino acids are independentlyselected from arginine and lysine. In other embodiments, the switch loopregion is modified to have three adjacent basic amino acids. In someembodiments, the native asparagine at position 296 is substituted forone of the three adjacent basic amino acids. The functional, modifiedRF1 protein is cleaved by OmpT1 at a faster rate than the wild-type RF1.For example, in one embodiment, the cleavage activity of modified RF1 byOmpT1 is greater than 50% of the wild type RF1 (SEQ ID NO:1) after 30minutes at 30° C. when the modified and wild-type proteins are presentat a similar concentration in a cell-free extract from bacteriaexpressing OmpT1.

In some embodiments, the functional RF1 that is cleavable by OmpT1contains an OmpT1 cleavage peptide in the switch loop region.

In a fourth aspect, the invention provides a nucleic acid encoding afunctional Releasing Factor 1 protein (RF1) that is cleavable by anOuter Membrane Protein T1 (OmpT1) where a scissile OmpT1 peptide bond islocated within the switch loop region of RF1 corresponding to aminoacids 287-304 of wild type RF1 (SEQ ID NO:1) and where the switch loopregion is modified to include two adjacent basic amino acids that arepositively charged at pH 7.0.

In a fifth aspect, the invention provides a method for reducing RF1competition at an amber codon in an in vitro cell free synthesis system,the method comprising the steps of:

-   -   i) culturing an OmpT1 positive bacteria expressing a functional        Releasing Factor 1 protein (RF1) that is cleavable by an Outer        Membrane Protein T1 (OmpT1)    -   ii) lysing the bacteria to create a cell free synthesis extract;    -   iii) contacting the RF1 protein in the extract with OmpT1 in an        amount to sufficient to reduce intact RF1 protein by 50%;    -   iv) adding a nucleic acid template to the extract where the        template codes for a protein of interest and includes an amber        codon; and,    -   v) allowing the cell free synthesis system to produce the        protein of interest,    -   where the RF1 protein has a scissile OmpT1 peptide bond is        located within the switch loop region corresponding to amino        acids 287-304 of wild type RF1 (SEQ ID NO:1) and where the        switch loop region is modified to include two adjacent basic        amino acids that are positively charged at pH 7.0.

In one embodiment of the method, the OmpT1 positive bacteria is from E.coli. In some embodiments, the oxidative phosphorylation system of thebacteria remains active after cell lysis and during the synthesis of theprotein of interest. In other embodiments, the cell free synthesissystem places a non-native amino acid at the amber codon of the proteinof interest.

In another aspect, the invention provides a cell free synthesis systemcomprising, in a single reaction mixture:

-   -   i) components from a bacterial lysate sufficient to translate a        nucleic acid template encoding a protein;    -   ii) a nucleic acid template encoding a protein of interest and        having at least one amber codon;    -   iii) tRNA complementary to the amber codon; and,    -   iv) a functional Releasing Factor 1 (RF1) protein cleavable by        an Outer Membrane Protein T1 (OmpT1), where a scissile OmpT1        peptide bond is located within the switch loop region of RF1        corresponding to amino acids 287-304 of wild type RF1 (SEQ ID        NO:1) and where the switch loop region is modified to include        two adjacent basic amino acids that are positively charged at pH        7.0.

In some embodiments, the cell free synthesis system reaction mixturefurther comprises a non-natural amino acid and corresponding amino acidsynthetase, the synthetase able to charge the tRNA complimentary to theamber codon with the non-natural amino acid. In some embodiments, thecell free synthesis system generates ATP via an active oxidativephosphorylation system.

In yet another aspect, the invention provides a method for expressingprotein in a cell-free synthesis system, comprising:

-   -   i. combining a nucleic acid template with a bacterial extract        comprising an RF1 protein that has been cleaved by OmpT1 at a        scissile OmpT1 peptide bond located within the switch loop        region corresponding to amino acids 287-303 of wild type RF1        (SEQ ID NO:1) and where the switch loop region has been modified        to include two adjacent basic amino acids that are positively        charged at pH 7.0 under conditions that permit protein        synthesis; and    -   ii. expressing a protein from the nucleic acid template.

In another aspect, the invention provides a bacterial cell comprising agenomically integrated sequence coding for a functional RF1 that iscleavable by OmpT1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that modified RF1 protein variants containing OmpT cleavagesites have wild-type RF1 activity when added to cell-free extractslacking OmpT activity. The ability of exogenous RF-1 variants to reducesuppression (increase chain termination) when adding a non-native aminoacid at an amber codon introduced at position S378 of the Fc protein(Fc_S378TAG) was determined. FIG. 1(A) Lane 1 is a control extract withno exogenously added RF1. The extract contains wild-type RF1, whichresults in reduced amber suppression, producing a truncated Fc (lowerband in gel). Lane 2 is a control extract containing exogenously addedwild-type (WT) RF1. The addition of wild-type RF1 results in a furtherreduction in amber suppression, which produces relatively more truncatedFc. Lanes 3-7 show cell-free extracts containing different RF1 variantsof the invention. All five variants possess RF1 activity. FIG. 1(B)shows the relative activity of RF1 variants, where added WT RF1 activityis set at 100 percent, and no added RF1 is set at zero percent.

FIG. 2 shows that cell-free extracts from a bacterial strain engineeredto express a modified RF1 variant (A18) produce a substantial increasein the yield of full-length IgG when nnAA were introduced at variouspositions of heavy chain, as compared to a bacterial strain expressing awild-type RF1. 2(A) left panel: SBJY001 extract, containing intact OmpTand wild-type RF1. 2(A) right panel: SBHS016 extract (strain 16),containing intact OmpT and RF1 variant A18. The band intensity offull-length (intact) IgG is stronger in strain 16 extract, indicatingthe incorporation of nnAA was substantially increased, with a decreasein truncated proteins. 2(B) shows the IgG soluble yield of proteinscontaining a nnAA at the indicated positions of heavy chain issubstantially increased in strain 16 extract, indicating that OmpTcleavage of RF1 variant A18 results in less truncated heavy chain.“RF1+” (RF1 positive) extract refers to RF1 from strain 001 that is notcleaved by OmpT. “RF1−” (RF1 negative) extract refers to RF1 from strain16 that is modified to include OmpT cleavage sites, and is thus degradedby OmpT activity.

FIG. 3 shows that cell-free extracts from a bacterial strain engineeredto express a modified RF1 variant (A18) produce a substantial increasein amber suppression corresponding to increased incorporation of nnAA atthe indicated positions of heavy chain. 3(A) left panel: SBJY001extract, containing intact OmpT and wild-type RF1. 3(A) right panel:SBHS016 (strain 16) extract, containing intact OmpT and RF1 variant A18.The band intensity of full-length (intact) heavy chain (Hc) is strongerin strain 16 extract, indicating the incorporation of nnAA wassubstantially increased, with a decrease in truncated proteins. 3(B)shows amber suppression of proteins containing a nnAA at the indicatedpositions of heavy chain is substantially increased in strain 16extract, indicating that OmpT cleavage of RF1 variant A18 results inincreased amber suppression. “RF1+” (RF1 positive) extract refers to RF1from strain 001 that is not cleaved by OmpT. “RF1−” (RF1 negative)extract refers to RF1 from strain 16 that is modified to include OmpTcleavage sites, and is thus degraded by OmpT activity.

DEFINITIONS

“Aminoacylation” or “aminoacylate” refers to the complete process inwhich a tRNA is “charged” with its correct amino acid that is a resultof adding an aminoacyl group to a compound. As it pertains to thisinvention, a tRNA that undergoes aminoacylation or has beenaminoacylated is one that has been charged with an amino acid, and anamino acid that undergoes aminoacylation or has been aminoacylated isone that has been charged to a tRNA molecule.

“Aminoacyl-tRNA synthetase” or “tRNA synthetase” or “synthetase” or“aaRS” or “RS” refers to an enzyme that catalyzes a covalent linkagebetween an amino acid and a tRNA molecule. This results in a “charged”tRNA molecule, which is a tRNA molecule that has its respective aminoacid attached via an ester bond.

“Codon” refers to a group of 3 consecutive nucleotides in a nucleic acidtemplate that specify a particular naturally occurring amino acid,non-native amino acid, or translation stop (polypeptide chaintermination) signal. Due to the degeneracy of the genetic code, an aminoacid can be specified by more than one codon.

An amber codon is a polypeptide chain-termination sequence (UAG) in RNA(TAG in DNA) that acts to terminate polypeptide translation in mostorganisms. The amber codon can also encode the proteinogenic amino acidpyrrolysine if the appropriate tRNA is charged by its cognateaminoacyl-tRNA synthetase.

“Amber codon tRNA” or “amber suppressor tRNA” or “amber anti-codon tRNA”refers to a tRNA that binds to an amber codon.

“Adjacent amino acids” refers to amino acids that immediately precede orfollow each other in the amino acid sequence of a polypeptide. Forexample, the amino acid at position two of a polypeptide's primary aminoacid sequence is adjacent to amino acids at positions one and three.“Two adjacent basic amino acids” refers to one basic amino acid thatimmediately precedes or follows another basic amino acid in the primaryamino acid sequence of a polypeptide. For example, the basic amino acidArg (R) at position 295 of the RF1 amino acid sequence can be adjacentto a basic amino acid such as K or R introduced at position 296 of theRF1 amino acid sequence.

“Bacterial derived cell free extract” refers to preparation of in vitroreaction mixtures able to translate mRNA into polypeptides. The mixturesinclude ribosomes, ATP, amino acids, and tRNAs. They may be deriveddirectly from lysed bacteria, from purified components or combinationsof both.

“Basic amino acids” are polar and positively charged at pH values belowtheir pK_(a)'s, and are very hydrophilic. Examples of basic amino acidsat neutral pH include Arginine (Arg=R), Lysine (Lys=K), and Histidine(His=H).

“Cell-free synthesis system” refers to the in vitro synthesis ofpolypeptides in a reaction mix comprising biological extracts and/ordefined reagents. The reaction mix will comprise a template forproduction of the macromolecule, e.g. DNA, mRNA, etc.; monomers for themacromolecule to be synthesized, e.g. amino acids, nucleotides, etc.;and co-factors, enzymes and other reagents that are necessary for thesynthesis, e.g. ribosomes, uncharged tRNAs, tRNAs charged with unnaturalamino acids, polymerases, transcriptional factors, etc.

The term “cleavage activity is greater than 50% of the wild type RF1”refers to the cleavage rate of a modified protein described herein beinggreater than 50% of the cleavage rate of wild-type protein underspecified conditions. For example, the cleavage rate can be greater than50% of the cleavage rate of wild-type protein after 30 minutes at 30° C.when the modified and wild-type proteins are present at a similarconcentration (e.g., a concentration of 0.1-1.0 micromolar) in acell-free extract from bacteria expressing OmpT1.

The term “deleterious activity” refers to an activity that decreases theyield of proteins during protein synthesis in cell free extracts. Forexample, the deleterious activity can inhibit cellular transcriptionand/or translation in cell free synthesis systems. The deleteriousactivity can include enzymatic reduction of GSSG that is required forefficient protein folding of disulfide bonded proteins produced in acell-free synthesis reaction. The deleterious activity can also includepremature chain termination by a releasing factor, such that polypeptideelongation is prematurely terminated at an introduced stop codon.

A “target protein” is a protein that has been modified to include aprotease cleavage site, or a protein that is modified to include a nnAA.An “essential target protein” is a protein that is required for normalgrowth and/or survival of a cell, such as a bacterial cell or eukaryoticcell.

A “functional Releasing Factor 1 (RF1) protein” refers to RF1 thatretains activity equal to or substantially similar to wild-type orunmodified RF1 protein. Functional RF1 activity can be tested, forexample, by measuring the growth rate of bacteria expressing themodified RF1 protein, and comparing the growth rate to bacteriaexpressing wild-type or unmodified RF1. The functional activity of otherproteins modified to contain OmpT cleavage sites can be similarlydetermined, for example, by measuring the growth rate of bacteriaexpressing the modified protein, and comparing the growth rate tobacteria expressing wild-type or unmodified protein. Functional RF1activity can also be tested, for example, by the ability of the modifiedRF1 protein to reduce orthogonal tRNA incorporation of a nnAA at aspecified position in an mRNA encoding a target protein, therebyincreasing the amount of premature chain termination (i.e., increasingthe amount of truncated protein).

The term “modified to include” in the context of the present inventionrefers to amino acid substitutions, additions, or deletions in a proteinor polypeptide sequence, or the corresponding substitutions, additions,or deletions in a nucleic acid sequence that encodes the modifiedprotein. The modified protein can include amino acid substitutions thatreplace an equal number of amino acids from the wild-type proteinsequence, amino acids added to the wild-type sequence, or amino acidsdeleted from the wild-type sequence. For example, the protein can bemodified to include basic amino acids that replace the wild-type aminoacid(s) at the corresponding position in the amino acid sequence. Theprotein can also be modified to include amino acid sequences that areknown protease cleavage sequences, such as OmpT cleavage sequences.

“Non-natural” or “non-native” amino acid refers to amino acids that arenot one of the twenty naturally occurring amino acids that are thebuilding blocks for all proteins that are nonetheless capable of beingbiologically engineered such that they are incorporated into proteins.Non-native amino acids may include D-peptide enantiomers or anypost-translational modifications of one of the twenty naturallyoccurring amino acids. A wide variety of non-native amino acids can beused in the methods of the invention. The non-native amino acid can bechosen based on desired characteristics of the non-native amino acid,e.g., function of the non-native amino acid, such as modifying proteinbiological properties such as toxicity, biodistribution, or half-life,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic properties, ability to react withother molecules (either covalently or noncovalently), or the like.Non-native amino acids that can be used in the methods of the inventionmay include, but are not limited to, an non-native analogue of atyrosine amino acid; an non-native analog of a glutamine amino acid; annon-native analog of a phenylalanine amino acid; an non-native analog ofa serine amino acid; an non-native analog of a threonine amino acid; analkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl,alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid,borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone,imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid,or any combination thereof; an amino acid with a photoactivatablecross-linker; a spin-labeled amino acid; a fluorescent amino acid; anamino acid with a novel functional group; an amino acid that covalentlyor noncovalently interacts with another molecule; a metal binding aminoacid; a metal-containing amino acid; a radioactive amino acid; aphotocaged and/or photoisomerizable amino acid; a biotin orbiotin-analog containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid; a chemically cleavable or photocleavable amino acid; anamino acid with an elongated side chain; an amino acid containing atoxic group; a sugar substituted amino acid, e.g., a sugar substitutedserine or the like; a carbon-linked sugar-containing amino acid, e.g., asugar substituted serine or the like; a carbon-linked sugar-containingamino acid; a redox-active amino acid; an alpha-hydroxy containing acid;an amino thio acid containing amino acid; an alpha,alpha-disubstitutedamino acid; a beta-amino acid; a cyclic amino acid other than praline,etc.

“Nucleic acid template” includes a DNA or RNA polynucleotide from whicha polypeptide will be translated. It will be understood by those ofskill in the art that a DNA nucleic acid template must first betranscribed into RNA, and that the RNA is translated into a polypeptide.DNA can be transcribed into RNA either in vivo or in vitro. The methodsof in vitro transcription of a DNA template are well known in the art.In some embodiments, the DNA template is subject to simultaneous invitro transcription and translation.

“Outer Membrane Protein T1” (OmpT1) is a surface membrane serineprotease and belongs to the omptin family of gram-negative bacteria.OmpT1 is known to cleave antimicrobial peptides, activate humanplasminogen, and degrade some heterologous recombinant proteins. OmpT1and its homologs cleave synthetic substrates between dibasic residueswith high catalytic efficiency. The cleavage of sequences containingdibasic residues has been shown to be important for the inactivation ofantibiotic peptides and colicins, the proteolysis of bacterial membraneproteins in trans, and the degradation of recombinant proteins expressedE. coli. In the context of this invention, OmpT1 has certain advantagesin that it is located on the outer membrane of the cell, and thus is notin contact with potential substrate proteins that are located inside theintact bacterial cell.

The term “oxidative phosphorylation system of the bacteria remainsactive” refers to a bacterial lysate that exhibits active oxidativephosphorylation during protein synthesis. For example, the bacteriallysate can generate ATP using ATP synthase enzymes and reduction ofoxygen. It will be understood that other translation systems known inthe art can also use an active oxidative phosphorylation during proteinsynthesis. The activation of oxidative phosphorylation can bedemonstrated by inhibition of the pathway using specific inhibitors,such as electron transport chain inhibitors.

“Translation” refers to the process whereby an RNA template is convertedinto a polypeptide containing natural and/or non-native amino acids, andis well known in the art. Translation involves the initiation step,whereby a ribosome attaches to the RNA template, generally at the FMetcodon (e.g., AUG), and the elongation step, whereby the anticodon of acharged tRNA molecule is paired with a codon in the RNA template, thisstep being repeated as the ribosome moves down the RNA template. As eachtRNA anticodon is paired with its corresponding codon, the amino groupof the amino acid charged to each tRNA molecule is covalently linked tothe carboxyl group of the preceding amino acid via peptide bonds.Generally, translation also involves the termination step, whereby theribosome encounters a translation stop codon, thus ending chainelongation and release of the polypeptide from the ribosome. However, inthe methods described herein, the RNA template can comprise an ORFhaving an amber stop codon UAG, which is recognized by the anti-codonCUA of a tRNA charged with a nnAA. Therefore, in the present methods,the amber codon does not necessarily function to terminate translation.

“Translation system” refers to a mixture of components that is able totranslate mRNA into polypeptides in vitro. A translation system can be acell free extract, reconstituted cell lysate, or a purified mixture ofcomponents that is able to translate mRNA into polypeptides in vitro.For example, the cell free extract or cell lysate used in the methodsdescribed herein can be derived from Escherichia coli (E. coli). Thetranslation system can also comprise a purified and reconstituted invitro translation system. The methods described herein can also utilizea translation system that exhibits active oxidative phosphorylationduring protein synthesis. For example, the translation system cangenerate ATP using ATP synthetase enzymes. The translation system canalso comprise at least one nucleic acid template. The nucleic acidtemplate can be simultaneously transcribed and translated into proteinusing the translation system.

“Reconstituted ribosomal translation system” refers to a mixture ofpurified components which is capable of translating a nucleic acidmolecule such as mRNA in vitro, as described in Tan et al., Methods 36,279-290 (2005), and Forster et al., U.S. Pat. No. 6,977,150, which areincorporated by reference herein in their entirety.

A “scissile OmpT1 peptide bond” is a peptide bond that is capable ofbeing cleaved by OmpT1.

A “surface exposed motif” is a domain that is present on the outside ofa protein molecule. Surface exposed motifs are generally more accessibleto proteolytic cleavage, for example by a serine protease such as OmpT1.One of skill in the art would understand that a surface exposed motifcan be defined in several ways. For example, a surface exposed motifoften lacks secondary structure, such as alpha helix or beta sheet.Further, surface exposed motifs often do not share a significant amountof amino acid sequence homology, even between homologous proteinsequences. However, one of skill in the art can calculate relativesurface accessible area for regions of a protein of interest using theGetArea algorithm embedded in pymol molecular modeling software (See theinternet at pymolwiki.org/index.php/Get_Area).

A “surface exposed motif having a B factor of at least 50 Å²” refers tothe mobility of individual atoms in the surface exposed motif Atoms insurface exposed motifs can have more mobility than atoms in the core ofa protein. B-factors generally indicate the relative vibrational motionof different parts of a structure, such as a protein. Atoms with lowB-factors belong to a part of the protein that is well-ordered and thushave relatively low mobility. Atoms with large B-factors generallybelong to part of the protein that is relatively flexible, and thus haverelatively high mobility. The higher the B-factor, the more likely thatpositional errors of an individual atom in a protein structure will beobserved. For example, a B factor of between 40 and 60 suggests thatpositional errors up to 1.0 Ångstrom can be observed. A B-factor ofgreater than 60 suggests that the atom is not likely to be within 1.0Ångstrom of its observed position. (See, e.g., the internet atwiki.cmbi.ru.nl/index.php/B-factor). The B-factor is calculated as:B _(i)=8π² U _(i) ²

where U_(i) ² is the mean square displacement of atom i. This produces aweighting factor on the contribution of atom i to the Fourier transformby:

$\exp\left( {{- B_{j}}\frac{\sin^{2}\Theta}{\lambda^{2}}} \right)$

As U increases, B increases and the contribution of the atom to thescattering is decreased. See, e.g., the internet at:

-   pldserver1.biochem.queensu.ca/˜rlc/work/teaching/definitions.shtml.    The B-factor is measured in units of Å².

The term “Ramachandran Plot” refers to a method for visualizing backbonedihedral angles ψ (Psi) versus φ (Phi) for each amino acid residue in aprotein structure. Methods for calculating Phi and Psi angles aredescribed, e.g., in Lovell, S. C. et al., Proteins: Structure, Function,and Genetics, 50:437-450 (2003), and Chen, V. B. et al., MolProbity:all-atom structure validation for macromolecular crystallography, ActaCrystallographica D66: 12-21 (2010). One can determine the Phi and Psiangles in a Ramachandran plot by referring to the above references, or,for convenience, one can use software programs freely available on theinternet. For example, one can upload a coordinate file or a PDB file tothe MOLPROBITY server at kinemage.biochem.duke.edu (see Chen, V. B. etal., supra). Alternatively, one can use the Ramachandran Plot Exploreravailable on the internet at boscoh.com/ramaplot.

The term “total solvent accessible surface area” (SASA) refers to thesurface area of a protein or polypeptide that is accessible to asolvent. Solvent accessible surface area can be described in units ofsquare ångstroms, and can be calculated using the ‘rolling ball’algorithm developed by Shrake & Rupley (Shrake, A; Rupley, J A. (1973).“Environment and exposure to solvent of protein atoms. Lysozyme andinsulin”. J Mol Biol 79 (2): 351-71.). Solvent accessible surface areacan also be calculated as described in Fraczkiewicz, R. and Braun, W.,“Exact and efficient analytical calculation of the accessible surfaceareas and their gradients for macromolecules,” J. Comp. Chem.,19:319-333 (1998). For convenience, one can calculate total solventaccessible surface area using software programs freely available on theinternet. For example, one can use the software routine GETAREA found atthe University of Texas Medical Branch websitecurie.utmb.edu/getarea.html to calculate solvent accessible surface area(solvation energy) of a protein molecule, as described in Fraczkiewicz,R. and Braun, W. (Id), by entering atomic coordinates in PDB (proteindatabase) format, and specifying the desired radius of the water probe(parameters: radius of the water probe=1.4).

The term “switch loop region” refers to a surface exposed motif thatlinks domains three and four of RF1 and forms a rigid connection thatplaces the GGQ motif of domain 3 in contact with the peptidyl-tRNA esterlinkage in the peptidyl transferase center (PTC) of the 50S subunit.Recognition of the stop codon by the termination complex stabilizes arearranged conformation of the switch loop region, which directs domain3 into the PTC. Rearrangement of the switch loop results inreorientation and extension of helix α7 so that the GGQ motifs docks inthe PTC. See, Korostelev, A. et al., “Recognition of the amber UAG stopcodon by release factor RF1,” EMBO Journal (2010) 29, 2577-2585. Theswitch loop region of RF1 corresponds to amino acids 287 to 304 of SEQID NO:1, and comprises the following amino acid sequence:287-QQAEASTRRNLLGSGDRS-304 (SEQ ID NO:4).

A “wild-type” protein is an unmodified protein having the amino acidsequence and/or functional activity of a native or naturally occurringprotein. For example, a wild-type RF1 protein can have the sequence ofSEQ ID NO:1. A “control protein” can be a wild-type protein or apreviously modified protein.

“Native amino acid” refers to one or more naturally occurring aminoacids encoded by the genetic code. An “endogenous native amino acid”refers to a native amino acid produced by the host cells used togenerate the lysate.

“Non-native amino acids” (“nnAA”) refers to chemical structures havingthe formula NH3-(CR)-COOH where R is not any of the 20 most commonsubstituents defining the natural amino acids.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same(e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%or 99% identity over a specified region), when compared and aligned formaximum correspondence over a comparison window, or designated region,as measured using the BLAST and PSI-BLAST algorithms, which aredescribed in Altschul et al. (J. Mol. Biol. 215:403-10, 1990), andAltschul, et al. (Nucleic Acids Res., 25:3389-3402, 1997), respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (see the internet atncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

When percentage of sequence identity is used in reference to apolypeptide, it is recognized that one or more residue positions thatare not otherwise identical can differ by a conservative amino acidsubstitution, in which a first amino acid residue is substituted foranother amino acid residue having similar chemical properties such as asimilar charge or hydrophobic or hydrophilic character and, therefore,does not change the functional properties of the polypeptide. Wherepolypeptide sequences differ in conservative substitutions, the percentsequence identity can be adjusted upwards to correct for theconservative nature of the substitution. Such an adjustment can be madeusing well-known methods, for example, scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions can be calculated using the algorithm described in Pearsonet al. (Meth. Mol. Biol. 24:307-331, 1994). Alignment also can beperformed by simple visual inspection and manual alignment of sequences.

The term “conservatively modified variation,” when used in reference toa particular polynucleotide sequence, refers to different polynucleotidesequences that encode identical or essentially identical amino acidsequences, or where the polynucleotide does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identicalpolynucleotides encode any given polypeptide. For instance, the codonsCGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.Thus, at every position where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons describedwithout altering the encoded polypeptide. Such nucleotide sequencevariations are “silent variations,” which can be considered a species of“conservatively modified variations.” As such, it will be recognizedthat each polynucleotide sequence disclosed herein as encoding a proteinvariant also describes every possible silent variation. It will also berecognized that each codon in a polynucleotide, except AUG, which isordinarily the only codon for methionine, and UUG, which is ordinarilythe only codon for tryptophan, can be modified to yield a functionallyidentical molecule by standard techniques. Accordingly, each silentvariation of a polynucleotide that does not change the sequence of theencoded polypeptide is implicitly described herein.

Furthermore, it will be recognized that individual substitutions,deletions or additions that alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 10%, andgenerally less than 1%) in an encoded sequence can be consideredconservatively modified variations, provided alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative amino acid substitutions providing functionally similaramino acids are well known in the art, including the following sixgroups, each of which contains amino acids that are consideredconservative substitutes for each another:

1) Alanine (Ala, A), Serine (Ser, S), Threonine (Thr, T);

2) Aspartic acid (Asp, D), Glutamic acid (Glu, E);

3) Asparagine (Asn, N), Glutamine (Gln, Q);

4) Arginine (Arg, R), Lysine (Lys, K)

5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Valine(Val, V); and

6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, W).

Two or more amino acid sequences or two or more nucleotide sequences areconsidered to be “substantially similar” if the amino acid sequences orthe nucleotide sequences share at least 50%, 60%, 70%, 80%, 90%, 95% or99% sequence identity with each other, or with a reference sequence overa given comparison window. Two or more proteins are also consideredsubstantially similar if they incorporate conservative amino acidsubstitutions providing functionally similar amino acids into the aminoacid sequence.

“Promoter” refers to sequence elements in the nucleic acid templatelocated upstream or downstream from the start of transcription. Promotersequences are involved in recognition and binding of RNA polymerase andother proteins that initiate transcription. Examples of promotersinclude T7, SP6 and T3 bacteriophage promoters.

“5′ untranslated region” refers to the nucleic acid sequence locatedupstream or 5′ of the open reading frame in a nucleic acid template. InRNA, the 5′ untranslated region precedes the translation start codonpresent in the nucleic acid template. In DNA, the 5′ untranslated regionrefers to nucleic acid sequences that are transcribed into RNA and arelocated 5′ to the translation start codon. In DNA, the translation startcodon is typically ATG, and in RNA the translation start codon istypically AUG.

“Primary amino acid sequence” refers to the order of amino acid residuesthat are joined together by peptide bonds into a polypeptide. The orderof amino acid residues is generally referenced starting at the aminoterminal end of the polypeptide and proceeding to the carboxy terminalend of the polypeptide. The primary amino acid sequence is determined bythe nucleotide sequence of RNA codons in the nucleic acid template.

“Open reading frame” refers to the nucleotide sequence of a nucleic acidtemplate that is translated into a polypeptide of interest. As usedherein, the open reading frame (ORF) can include at least one codoncorresponding to a defined amino acid residue that binds to a tRNAcharged with a nnAA. The at least one codon can be an amber codon.

“Isoaccepting sense tRNA” refers to different tRNA species that bind toalternate codons for the same amino acid.

“tRNA” or “transfer RNA” refers to a small RNA molecule that transfers aspecific amino acid to a growing polypeptide chain at the ribosomal siteof protein synthesis during translation. tRNAs contain a three basecodon that pairs to the corresponding mRNA codon. As a result of thedegeneracy of the genetic code, an amino acid can associate withmultiple tRNAs, while each type of tRNA molecule can only associate withone type of amino acid.

The terms “polypeptide” and “protein” are used interchangeably, andrefer to a compound containing two or more amino acids joined by peptidebonds. Proteins can contain one or more polypeptide chains.

The term “when the protein is uncomplexed” refers to the structure of aprotein in solution, or a portion, region or domain of the structure ofthe protein in solution, and not the structure of the protein, orportion, region or domain of the structure of the protein, when it ispart of a complex with other molecules such as proteins, ligands, enzymesubstrate or inhibitor complexes, ribosomes, bound antibodies orantibody fragments, RNA, and DNA. The term also refers to the structureof a protein domain, such as the amino acids comprising a surfaceexposed motif, that is in solution.

As used herein, the term “about,” when modifying any amount, refers tothe variation in that amount typically encountered by one of skill inthe art, e.g., in protein synthesis or X-ray crystallographyexperiments. For example, the term “about” refers to the normalvariation encountered in measurements for a given analytical technique,both within and between batches or samples. Thus, the term about caninclude variation of 1-10% of the measured value, such as 5% or 10%variation. The amounts disclosed herein include equivalents to thoseamounts, including amounts modified or not modified by the term “about.”

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention provides target proteins that are recombinantlymodified to include OmpT1 protease cleavage sites that are capable ofbeing cleaved by OmpT1. The OmpT1 cleavage site includes a scissileOmpT1 peptide bond. In one embodiment, the target protein is modified toinclude two adjacent basic amino acids that are positively charged at pH7.0. In some embodiments, the target protein is modified to introducethe OmpT1 cleavage site into a surface exposed motif of the targetprotein. In some embodiments, the scissile OmpT1 peptide bond is locatedin a surface exposed motif having a B factor of at least 50 Å² when theprotein or the surface exposed motif is uncomplexed (i.e., free insolution and/or not part of a macromolecular structure comprising othermolecules). In some embodiments, the scissile OmpT1 peptide bond islocated in a surface exposed motif having a total solvent accessiblesurface area (SASA) of between about 25 Å² and about 225 Å². In someembodiments, the target protein is modified such that the scissile OmpT1peptide bond is located in a position of the protein which exhibits aPhi angle of from 0° to −180° or a Psi angle from 0° to +180° in aRamachadran Plot.

As is understood by one of skill in the art, the above embodimentsrequire structural information for the protein domain(s) of interest.However, surface loops often are disordered, and therefore no crystalstructure may be available for the target protein of interest. Thus, insome embodiments, a homology model can be made using similar structuresto predict a SASA of between about 25 Å² and about 225 Å², or a Phiangle of from 0° to −180° or a Psi angle from 0° to +180° in aRamachadran Plot, in the absence of structural information.

The invention further provides a bacterial cell that express both OmpT1and the recombinantly modified target protein. In some embodiments, therecombinantly modified target protein is an essential protein, forexample, a protein that is required for normal cell growth and/orsurvival.

The target proteins described herein are selected because they havedeleterious activity in an in vitro cell-free synthesis system. Forexample, the target protein can inhibit transcription and/or translationof nucleic acid templates that encode proteins of interest. Thus, theinvention provides methods for reducing the deleterious activity of amodified essential target protein in an in vitro cell-free synthesissystem by inactivating the target protein with an OmpT1 protease. Insome embodiments, the method comprises culturing an OmpT1 positivebacteria expressing the modified essential target protein, where thetarget protein is modified to include an OmpT1 cleavage site comprisinga scissile OmpT1 peptide bond in a surface exposed motif; lysing thebacteria to create a cell free synthesis extract; contacting themodified essential target protein with OmpT1 in an amount sufficient toreduce the amount of intact target protein by 50%; adding a nucleic acidtemplate to the extract, where the template codes for a protein ofinterest; and allowing the cell free synthesis system to produce theprotein of interest. The method takes advantage of the spatialseparation of the modified target protein and the OmpT1 protease duringcell growth, when the target protein may be important for cell growthand/or survival, whereas the target protein is cleaved by OmpT1 when thebacterial cells are disrupted and lysed to produce a cell-free extract.

In some embodiments of the method, the scissile OmptT1 peptide bond islocated within a surface exposed motif having a B factor of at least 50Å² when the protein or the surface exposed motif is uncomplexed. Forexample, the surface exposed motif can have a B factor of 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 Å² or greater, or can be in a range of between about 50 andabout 200 Å², between about 50 and about 150 Å², between about 50 andabout 100 Å², between about 60 and about 200 Å², between about 60 andabout 150 Å², or between about 60 and about 100 Å². In some embodimentsof the method, the scissile OmptT1 peptide bond is located within asurface exposed motif having a total solvent accessible surface area ofbetween about 25 Å² and about 225 Å². For example, in some embodiments,the total solvent accessible surface area is about 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, or 225 Å² (where the term “about”modifies each of the preceding values). In one embodiment of the method,the scissile OmptT1 peptide bond is located in a position of the proteinwhich exhibits a Phi angle of from 0° to −180° or a Psi angle from 0° to+180° in a Ramachadran Plot. As described above, in the absence ofstructural information, a homology model can be made using similarstructures to predict a SASA of between about 25 Å² and about 225 Å², ora Phi angle of from 0° to −180° or a Psi angle from 0° to +180° in aRamachadran Plot.

The proteins of interest referred to above include proteins that areengineered to incorporate non-native amino acids (nnAA) at a definedlocation of the amino acid sequence. The introduction of nnAA intoproteins can result in proteins with preferred properties. One methodfor introducing nnAA into proteins or polypeptides of interest usesaminoacylated orthogonal tRNAs that recognize an amber (stop) codon forintroducing the nnAA into the nascent polypeptide chain during proteintranslation. However, the yield of proteins having nnAA introducedtherein can be decreased by proteins of the translation terminationcomplex that facilitate the termination of translation by recognizingthe termination or stop codons in an mRNA sequence. Release Factor 1(RF1) is part of the termination complex, and recognizes the UAG (amber)stop codon. RF1 recognition of the amber codon can promote pre-maturechain termination at the site of nnAA incorporation, which reduces theyield of desired proteins. The methods described herein solve thisproblem by decreasing the functional activity of RF1 in bacterial celllysates. Thus, in some embodiments, the essential target protein is RF1.The functional activity of RF1 is decreased by introducing OmpT1protease cleavage sites into RF1. OmpT1 is an enzyme located on theouter cell membrane of intact bacteria. Thus, the modified RF1 is notavailable as a substrate for OmpT1 in intact cells. When the bacterialmembrane is disrupted, for example, as in a bacterial lysate, the OmpT1enzyme is able to contact and cleave the modified RF1 at the introducedcleavage site, thereby decreasing the functional activity of RF1. Themethods are applicable to other releasing factors found in thetranslation termination complex, such as RF2, which recognizes the UGAstop codon. The methods are also applicable to proteins that degrademRNA during in vitro translation, such as RNase enzymes. Further, themethods are generally applicable to any protein in a bacterial extractthat inhibits transcription and/or translation of a protein of interest.

General Methods

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. Practitioners are particularly directedto Green, M. R., and Sambrook, J., eds., Molecular Cloning: A LaboratoryManual, 4th ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2012), and Ausubel, F. M., et al., Current Protocols inMolecular Biology (Supplement 99), John Wiley & Sons, New York (2012),which are incorporated herein by reference, for definitions and terms ofthe art. Standard methods also appear in Bindereif, Schón, & Westhof(2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany whichdescribes detailed methods for RNA manipulation and analysis, and isincorporated herein by reference. Examples of appropriate moleculartechniques for generating recombinant nucleic acids, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Green, M. R., and Sambrook, J., (Id.); Ausubel, F. M., et al.,(Id.); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif. 1987);and PCR Protocols: A Guide to Methods and Applications (Academic Press,San Diego, Calif. 1990), which are incorporated by reference herein.

Methods for protein purification, chromatography, electrophoresis,centrifugation, and crystallization are described in Coligan et al.(2000) Current Protocols in Protein Science, Vol. 1, John Wiley andSons, Inc., New York. Methods for cell-free synthesis are described inSpirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim,Germany. Methods for incorporation of non-native amino acids intoproteins using cell-free synthesis are described in Shimizu et al (2006)FEBS Journal, 273, 4133-4140.

PCR amplification methods are well known in the art and are described,for example, in Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press Inc. San Diego, Calif., 1990. Anamplification reaction typically includes the DNA that is to beamplified, a thermostable DNA polymerase, two oligonucleotide primers,deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.Typically a desirable number of thermal cycles is between 1 and 25.Methods for primer design and optimization of PCR conditions are wellknown in the art and can be found in standard molecular biology textssuch as Ausubel et al., Short Protocols in Molecular Biology, 5^(th)Edition, Wiley, 2002, and Innis et al., PCR Protocols, Academic Press,1990. Computer programs are useful in the design of primers with therequired specificity and optimal amplification properties (e.g., OligoVersion 5.0 (National Biosciences)). In some embodiments, the PCRprimers may additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto specific restriction enzyme sites in a vector. If restriction sitesare to be added to the 5′ end of the PCR primers, it is preferable toinclude a few (e.g., two or three) extra 5′ bases to allow moreefficient cleavage by the enzyme. In some embodiments, the PCR primersmay also contain an RNA polymerase promoter site, such as T7 or SP6, toallow for subsequent in vitro transcription. Methods for in vitrotranscription are well known to those of skill in the art (see, e.g.,Van Gelder et al., Proc. Natl. Acad. Sci. U.S.A. 87:1663-1667, 1990;Eberwine et al., Proc. Natl. Acad. Sci. U.S.A. 89:3010-3014, 1992).

OmpT1 Cleavable Proteins

The present invention provides target proteins that are capable of beingcleaved and inactivated by proteolytic enzymes such as OmpT1. In orderto be cleaved by the protease, the proteins are modified to introduceproteolytic cleavages sites into the protein. In some embodiments, thecleavage site introduced into the modified target protein is a scissileOmpT1 peptide bond, and the scissile OmpT1 is introduced into a surfaceexposed motif of the target protein. The cleavage site recognized byOmpT1 comprises two adjacent basic residues. Thus, the target proteinsdescribed herein are modified to comprise two adjacent basic residuesthat are positively charged at pH 7.0.

Modification of Proteins to Introduce an OmpT1 Cleavage Site in aSurface Exposed Motif Having a B Factor of at Least 50 Å²

In some embodiments, the target protein is modified to introduce thescissile OmpT1 peptide bond into a surface exposed motif having a Bfactor of at least 50 Å² when the amino acid sequence of the targetprotein or motif region is uncomplexed. The location of a surfaceexposed motif having a B factor of at least 50 Å² can be determined inseveral ways. B-factors are given for each atom in crystallographicProtein Data Bank (PDB) files. The B factor of a surface exposed motifcan be calculated using the GetArea algorithm embedded in pymolmolecular modeling software (See the internet atpymolwiki.org/index.php/Get_Area). Alternatively, if an X-raycrystallographic structure of the protein is not available, but an NMRstructure of the protein is available, the random coil index (RCI) maybe used in place of a large B-factor (See Berjanskii, M. V., et al.,Application of the random coil index to studying protein flexibility, JBiomol NMR. 2008 January;40(1):31-48. Epub 2007 Nov. 6).

In order to modify a target protein to introduce an OmpT1 cleavage siteinto a surface exposed motif having a B factor of at least 50 Å², theB-factors from X-ray crystallographic structures of the protein can bemapped to the amino acid sequence, and the sequence recombinantlymodified to include a scissile OmptT1 peptide bond at site(s) with largeB-factors. The recombinantly modified target protein can then be testedfor degradation of the protein in a cell-extract containing OmpT1. Insome embodiments, the B factor is at least 50, 60, 70, 80, 90 or 100 Å².In some embodiments, the B factor is between about 50 and about 200 Å²,or between about 50 and about 150 Å², or between about 50 and 120 Å². Itis understood that ranges described herein include all values in betweenthe end points.

Modification of Proteins to Introduce an OmpT1 Cleavage Site in aSurface Exposed Motif Having a Total Solvent Accessible Surface Area ofBetween About 25 Å² and About 225 Å².

In some embodiments, the target protein is modified to introduce thescissile OmpT1 peptide bond into a surface exposed motif having a totalsolvent accessible surface area of between about 25 Å² and about 225 Å².The location of a surface exposed motif having a total solventaccessible surface area of between about 25 Å² and about 225 Å² can bedetermined using methods known in the art. For example, the softwareroutine GETAREA can be used to locate solvent-exposed vertices ofintersecting atoms, as described in Fraczkiewicz, R. and Braun, W.,“Exact and efficient analytical calculation of the accessible surfaceareas and their gradients for macromolecules,” J. Comp. Chem., 19,319-333 (1998). Thus, one can calculate solvent accessible surface area(solvation energy) of a protein molecule using the GETAREA software byentering atomic coordinates in PDB (protein database) format, specifyingthe desired radius of the water probe, and specifying the desired levelof output using a form provided on the internet at the University ofTexas Medical Branch website curie.utmb.edu/getarea.html (parameters:radius of the water probe=1.4). Other methods for determining totalsolvent accessible surface area are described in Eisenberg, D. andMcLachlan, A. D. (1986) Nature, 319, 199; Markley, J. L.; et al. (1998)Pure & Appl. Chem., 70, 117; and Wesson, L. and Eisenberg, D. (1992)Protein Sci., 1, 227.

Modification of Proteins to Introduce an OmpT1 Cleavage Site in aPosition of the Protein which Exhibits a Phi Angle of from 0° to −180°or a Psi Angle from 0° to +180° in a Ramachadran Plot

In some embodiments, the target protein is modified to introduce thescissile OmpT1 peptide bond in a position of the protein which exhibitsa Phi angle of from 0° to −180° or a Psi angle from 0° to +180° in aRamachadran Plot. The location of the position in a protein whichexhibits a Phi angle of from 0° to −180° or a Psi angle from 0° to +180°in a Ramachadran Plot can be determined using methods known in the art.Methods for calculating Phi and Psi angles are described, e.g., inLovell, S. C. et al., Proteins: Structure, Function, and Genetics,50:437-450 (2003). One can determine the Phi and Psi angles in aRamachandran plot by uploading a coordinate file or a PDB file on theMOLPROBITY server on the internet at kinemage.biochem.duke.edu (seeChen, V. B., et al., (2010) MolProbity: all-atom structure validationfor macromolecular crystallography. Acta Crystallographica D66: 12-21.).Alternatively, one can use the Ramachandran Plot Explorer available onthe internet at boscoh.com/ramaplot.

The modified proteins described herein are selected because they caninhibit production of proteins in cell-free synthesis systems. Thus, insome embodiments, the modified proteins described herein decrease theproduction of full length proteins in bacterial cell-free extracts. Forexample, the proteins RF1 and RF2 are part of the termination complexthat recognizes a stop codon in the mRNA and terminates translation ofthe polypeptide chain. Premature termination of translation isundesirable when incorporating non-native amino acids into a proteinusing cell free translation systems as described herein. Reducingrecognition of the stop codon by the termination complex can increasethe yield of proteins incorporating nnAA. Thus, in some embodiments, theRF1 and/or RF2 protein, or a function homolog thereof, is modified tocontain OmpT1 cleavage sites in a surface exposed motif having a BFactor of at least 50 Å² when the RF1 and/or RF2 protein, or functionalhomolog thereof, is uncomplexed. In some embodiments, the RF1 and/or RF2protein, or a function homolog thereof, is modified to contain OmpT1cleavage sites in a surface exposed motif having a total solventaccessible surface area of between about 25 Å² and about 225 Å². In someembodiments, the RF1 and/or RF2 protein, or a function homolog thereof,is modified to contain OmpT1 cleavage sites in a position of the proteinwhich exhibits a Phi angle of from 0° to −180° or a Psi angle from 0° to+180° in a Ramachadran Plot. In some embodiments, the functionalmodified protein is substantially similar to RF1 (SEQ ID NO:1) or RF2(SEQ ID NO:2).

The RF1 protein includes the RF1 wildtype prototype protein (SEQ IDNO:1) from E. coli as well as polymorphic variations and recombinantlycreated muteins. RF1 proteins are defined as having substantially thesame biological activity or functional capacity as the wild type (e.g.,at least 80% of either), have at least 60%, 70%, 80%, 90% or 95%sequence identity to the prototype protein RF1, and/or bind topolyclonal antibodies generated against the prototype protein SEQ IDNO:1.

With regard to the binding of an RF1 protein to polyclonal antibodies,the RF1 protein will bind under designated immunoassay conditions to thespecified antibodies with a specificity at least two times thebackground, where the antibodies do not substantially bind in asignificant amount to other proteins present in the sample. For example,polyclonal antibodies raised to RF1 (SEQ ID NO:1), or isoforms orportions thereof, can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with RF1 and not withother proteins, except for polymorphic variants of RF1. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

As described in the Examples, RF1 was successfully modified to introducefunctional OmpT1 cleavage sites in a surface exposed motif. However, theintroduction of putative dibasic OmpT1 cleavage sites into RF1 did notalways result in a protein that could be efficiently cleaved by OmpT1.For example, the single amino acid substitutions M74R, E76K, E84K, A85R,E87R, E108R, T293R and S304K were introduced into the loop regions ofRF1 beside an existing Arg or Lys, thereby creating dibasic cleavagesites. However, these variants were not efficiently cleaved when the RF1variants were expressed in OmpT positive cell extracts. The presentinvention therefore provides the unexpected result that proteins such asRF1 can be modified such that the protein is capable of being cleaved bya protease such as OmpT1.

As will be understood by persons of skill in the art, the introducedcleavage site can be cleaved by an enzyme with OmpT1-like enzymeactivity, where the enzyme activity results from a functional homolog orfragment of OmpT1, or from a protein that is modified to have OmpT1-likeactivity.

Cleavage of Unmodified Proteins by OmpT1

The proteins described herein can also contain endogenous OmpT1 proteasecleavage sites. For example, in some embodiments, the unmodified orwild-type protein contains a dibasic amino acid sequence comprising ascissile OmpT1 peptide bond that is cleavable by OmpT1. Cleavage of theprotein by OmpT1 can be tested by incubating the purified protein withbacterial cell-free extracts that express OmpT1, and comparing theamount of cleavage therein with the amount of cleavage detected incell-free extracts that do not express OmpT1. In some embodiments, theunmodified proteins containing endogenous OmpT1 cleavage sites arenecessary or required for normal cell growth or function, but inhibitthe translation of proteins in cell-free extracts. Thus, the inventionprovides methods for selectively inactivating unmodified proteins bycleavage with OmpT1, where the timing of inactivation can be controlledsuch that the proteins are functional during cell growth, but areinactivated in cell-extracts expressing OmpT1.

Template

In order to produce the proteins of this invention, one needs a nucleicacid template. Templates for the invention are used to produce theproteins modified to comprise a scissile OmpT1 cleavage site. Templatesfor the invention are also used to produce proteins of interest that areexpressed in cell-free systems. The templates for cell-free proteinsynthesis can be either mRNA or DNA. The template can comprise sequencesfor any particular gene of interest, and may encode a full-lengthpolypeptide or a fragment of any length thereof. Nucleic acids thatserve as protein synthesis templates are optionally derived from anatural source or they can be synthetic or recombinant. For example,DNAs can be recombinant DNAs, e.g., plasmids, viruses or the like.

In one embodiment, the DNA template comprises an ORF encoding a targetprotein comprising an OmpT1 cleavage site. For example, the ORF canencode a modified target protein that is required for normal cell growthand survival, but whose activity decreases the yield of proteins incell-free extracts, either directly or indirectly. Thus, the ORF mayencode a protein component of the termination complex, such as RF1 orRF2 or modified variants thereof. The ORF may also encode an RNase thatdegrades RNA templates.

In another embodiment, the DNA template comprises an ORF encoding aprotein of interest that is modified to incorporate a non-native aminoacid. Thus, the ORF may encode any protein having biological importancethat incorporates a nnAA at a defined position of the amino acidsequence. Non-limiting examples of such proteins of interest includeantibodies, hormones, cytokines, and viral proteins. The invention usessense codons for the incorporation of non-native amino acids, andcircumvents the requirement of orthogonal components as is commonlyfound in the art. In these embodiments, the ORF comprises at least oneisoaccepting sense codon. The ORF further comprises one codoncorresponding to a defined amino acid residue that recognizes a tRNAcharged with a non-native amino acid. In some embodiments, the ORFcomprises an amber codon (UAG) that binds a tRNA charged with anon-native amino acid. In other embodiments, the template is capable oftranslating a complete and functional protein regardless of whethernon-native amino acids are chosen to be incorporated into the protein ofinterest.

A DNA template that comprises the ORF of interest will be operablylinked to at least one promoter and to one or more other regulatorysequences including without limitation repressors, activators,transcription and translation enhancers, DNA-binding proteins, etc.Suitable quantities of DNA template for use herein can be produced byamplifying the DNA in well known cloning vectors and hosts, or bypolymerase chain reaction (PCR).

The DNA template can further comprise the ORF of interest joined inframe to nucleic acid sequences that encode amino acid sequences thatare useful for isolating and purifying the expressed protein, such aspoly-amino acid tags that bind with high affinity to chromatographymedia. The poly-amino acid tag can be located at the 5′ end or 3′ end ofthe ORF, resulting in an amino-terminal or carboxyl terminal tag in theexpressed protein, respectively. In one embodiment, the ORF is joined inframe to sequences that encode a poly-Histidine tag.

One embodiment uses a bacterial lysate. A DNA template can beconstructed for bacterial expression by operably linking a desiredprotein-encoding DNA to both a promoter sequence and a bacterialribosome binding site (Shine-Delgarno sequence). Promoters suitable foruse with the DNA template in the cell-free transcription-translationmethods of the invention include any DNA sequence capable of promotingtranscription in vivo in the bacteria from which the bacterial extractis derived. Preferred are promoters that are capable of efficientinitiation of transcription within the host cell. DNA encoding thedesired protein and DNA containing the desired promoter andShine-Dalgarno (SD) sequences can be prepared by a variety of methodsknown in the art. Alternatively, the desired DNA sequences can beobtained from existing clones or, if none are available, by screeningDNA libraries and constructing the desired DNA sequences from thelibrary clones.

RNA templates encoding the protein of interest can be convenientlyproduced from a recombinant host cell transformed with a vectorconstructed to express a mRNA with a bacterial ribosome binding site (SDsequence) operably linked to the coding sequence of the desired genesuch that the ribosomes in the reaction mixture are capable of bindingto and translating such mRNA. Thus, the vector carries any promotercapable of promoting the transcription of DNA in the particular hostcell used for RNA template synthesis.

Because it is difficult to extract un-degraded RNA from bacteria, highereukaryotic cell culture is preferred for the production of the RNAtemplate. In principle, any higher eukaryotic cell culture is workable,including both vertebrate and invertebrate cell cultures. The RNAtemplate can be conveniently isolated in a total cellular RNA fractionextracted from the host cell culture. Total cellular RNA can be isolatedfrom the host cell culture by any method known in the art. The desiredRNA template can be isolated along with most of the cellular mRNA if theRNA template is designed to contain at its 3′ end a polyadenylationsignal recognized by the eukaryotic host cell. Thus, the host cell willproduce the RNA template with a polyadenylate (poly(A)) tail.Polyadenylated mRNAs can be separated from the bulk of cellular RNA byaffinity chromatography on oligodeoxythymidylate (oligo (dT))-cellulosecolumns using any methods known in the art. If the size of the mRNAencoding the desired protein is known, the mRNA preparation can befurther purified for mRNA molecules of the particular size by agarosegel electrophoresis of the RNA.

Expression of Modified Proteins Having OmpT1 Cleavage Sites

Once the nucleic acid template is produced, the template is used toexpress the recombinant target protein comprising an OmpT1 cleavage sitein a cell, or to synthesize a modified recombinant target protein in acell-free translation system. For example, the template can be added toa cell lysate under conditions sufficient to translate the template intoprotein. The cell lysate can be from bacterial cells or eukaryoticcells. The expressed protein can then be purified using methods known inthe art, as described below.

Purifying Proteins to Test for Activity

Proteins containing OmpT1 cleavage sites can be purified as is standardin the art. Proteins of the invention can be recovered and purified bymethods including, but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis, etc. For example, proteins comprising N-terminalHistidine tags can be purified using affinity media containing metalions such as nickel and cobalt. The affinity media is then washed toremove unbound protein, and the bound proteins eluted and recovered. Insome embodiments, the poly-histidine tagged proteins are purified usingImmobilized Metal Affinity Chromatography (IMAC). The modified proteinscan also be purified using high performance liquid chromatography(HPLC), or other suitable methods where high purity is desired. Apreferred purification method is provided in Example 1.

Following purification, proteins containing OmpT1 cleavage sites canpossess a conformation different from the desired conformations of therelevant polypeptides. Thus, the purified proteins can be subjected toconditions that result in the preferred protein conformation. A varietyof purification/protein folding methods are known in the art, e.g.,Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification(Academic Press, Inc. N.Y. 1990); Bollag et al., Protein Methods, 2ndEdition, (Wiley-Liss, N.Y. 1996). In general, it is occasionallydesirable to denature and reduce expressed polypeptides and then tocause the polypeptides to re-fold into the preferred conformation. Forexample, guanidine, urea, DTT, DTE, and/or a chaperone can be added to atranslation product of interest. Methods of reducing, denaturing andrenaturing proteins are well known to those of skill in the art. See,e.g. Debinski et al., J. Biol. Chem. 268:14065-70 (1993); Buchner etal., Anal. Biochem. 205:263-70 (1992).

Confirmation of Functional Biological Activity of Modified ProteinsHaving OmpT1 Cleavage Sites

Depending on the desired use of the modified proteins described herein,it can be important for the modified proteins to have biologicalactivity comparable to an unmodified, wild-type protein. For example, ifthe protein to be modified is important for normal growth of bacteria,it is desirable to retain the normal activity levels of the proteinuntil the cells are lysed to produce the cell-free lysate fortranslation. Thus, the methods of the present invention provide formodified proteins containing OmpT1 cleavage sites that have biologicalactivity comparable to the native or wild-type protein. Modifiedproteins that retain wild-type levels of activity, or activity levelsthat are comparable to wild-type, are referred to herein as a functionalproteins. One may determine the specific activity of a protein bydetermining the level of activity in a functional assay. Alternatively,one may determine the specific activity of a protein by quantitating theamount of protein present in a non-functional assay, e.g.immunostaining, ELISA, quantitation on coomasie or silver stained gel,etc., and determining the ratio of biologically active protein to totalprotein. Generally, a modified protein is comparable to a wild-typeprotein if the specific activity as thus defined is at least about 50%of the wild-type protein, or at least about 60%, about 70%, about 80%,about 90% or greater than that of the wild-type protein. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 1989).

The functional activity of modified proteins can be tested in a varietyof ways. For example, the growth rate of bacteria expressing themodified protein can be compared to the growth rate of bacteriaexpressing a wild-type, control or unmodified protein. The functionalactivity of modified proteins can also be tested by determining theability of the modified protein to terminate translation at a stop codonin the mRNA template. Termination of translation results in truncatedproteins, and thus the relative activities of modified proteins can bequantitated by measuring the amount of truncated and full lengthprotein, and comparing the ratio of truncated to full length proteinwith that resulting from wild-type protein, as described in the Examplesand shown in FIG. 1. A modified protein has comparable activity to awild-type protein if the ratio of truncated to full length protein is atleast about 50%, 60%, 70%, 80%, 90%, 95%, or greater than the ratio oftruncated to full length protein using a wild-type protein.

Confirmation that Modified Proteins are Cleavable by OmpT1

Once the modified proteins are determined to have comparable biologicalactivity to the wild-type or unmodified protein, the modified proteinsare tested to determine that they are cleaved by OmpT1. One method fortesting that the modified proteins of the invention are cleaved by OmpT1is to add recombinant protein containing OmpT1 cleavage sites to acell-free extract containing functional OmpT1. If the modified proteinis cleaved by OmpT1, it will migrate at an apparent lower molecularweight than the intact protein during gel electrophoresis (e.g.,SDS-PAGE). The modified protein can be detected by including aradioactive label such as ¹⁴C in the translation reaction underconditions suitable for incorporation of the radioactive label into themodified protein. The migration of the radioactively labeled protein canbe visualized, for example, on an autoradiograph of the gel. Cleavage ofthe modified protein by OmpT1 can also be detected by Western blotanalysis, for example, by transferring the proteins from the gel to asolid support, contacting the support with antibodies that bind to theintact and cleaved protein, and visualizing the bound antibodies using adetectable label.

The cleavage activity by OmpT1 can be determined by comparing the amountor rate of cleavage of a modified protein by OmpT1 to that of anunmodified protein. For example, the cleavage rate of the modifiedprotein by OmpT1 can be greater than 50% of the cleavage rate of thewild-type protein under specified conditions. In some embodiments, thecleavage rate of a modified protein described herein is greater than 50%of the cleavage rate of wild-type protein after 30 minutes at 30° C.when the modified and wild-type proteins are present at a similarconcentration (e.g., a concentration of 0.1-1.0 micromolar) in acell-free extract from bacteria expressing OmpT1. In some embodiments,greater than 90% of the modified protein is cleaved by OmpT1 after 60minutes at 30° C. when the modified protein is present at aconcentration of 0.1-1.0 micromolar in a cell-free extract from bacteriaexpressing OmpT1.

It will be understood that the OmpT cleavage sites introduced into themodified proteins described herein are cleavable by any protein orpolypeptide that possesses OmpT-like enzyme activity. All that isrequired is that the OmpT1-like enzyme be capable of cleaving a proteinmodified to contain OmpT cleavage sites. For example, the OmpT-likeproteolytic activity can be provided by wild-type or native OmpT1, or afunctional homolog or fragment thereof. The OmpT-like activity can alsobe provided by a protein that is substantially identical orsubstantially similar to OmpT1. For example, the protein with OmpT-likeactivity can have at least 60%, 70%, 80%, 90%, 95% or 99% sequenceidentity to OmpT1. In some embodiments, the protein having OmpT-likeactivity is at least 60%, 70%, 80%, 90%, 95% or 99% identical to SEQ IDNO:3. In some embodiments, the protein having OmpT1 activity isspecifically bound by a polyclonal antibody that binds to OmpT1 andvariants thereof. Selection of polyclonal antibodies that bind to OmpT1and functional variants thereof can be performed as described herein forselecting polyclonal antibodies that bind RF1.

Transforming Bacteria with the OmpT1 Cleavable Proteins

Once the modified essential target proteins are determined to retainwild-type function and to be susceptible to cleavage by OmpT1, asdescribed above, nucleic acids encoding the modified proteins aretransformed into bacteria. The bacteria can be transformed with thenucleic acid under conditions suitable for incorporation of the nucleicacid into the genome of the bacteria. For example, the bacteria can betransformed with oligonucleotides having sequences that encode the OmpT1cleavage sites described herein using oligonucleotide-mediated allelicreplacement, as described in the Examples. Incorporation of the desiredmutations in the bacterial genome can be determined, for example, byscreening transformed colonies using Mismatch Amplification MutationAssay (MAMA) PCR, as described in the Examples.

Use of OmpT1 Cleavable Proteins to Increase the Yield of Proteins inCell-Free Translation Systems

The modified target proteins having OmpT1 cleavage sites are efficientlydegraded when expressed in a bacterial cell extract that contains activeOmpT1 protease. Among the various uses of this invention, carefulselection of inhibitory proteins that are modified to be cleavable byOmpT1 can enhance the productivity of cell-free synthesis systems. Forexample, in one preferred use, the cleavage of selected target proteinsby OmpT1 can improve the translation efficiency of full length proteinsincorporating non-native amino acids in cell-free extracts. In certainembodiments, the non-native amino acid is incorporated at an amber codonintroduced into the mRNA template. As described above, the incorporationof non-native amino acids at an amber codon can be inhibited bytermination complex proteins such as RF1 and RF2. Thus, in aparticularly desirable embodiment, cleavage and inactivation of RF1and/or RF2 by OmpT1 can increase the yield of proteins engineered toincorporate a non-native amino acid at an amber codon.

Non-Native Amino Acids

As described above, in one preferred use, degradation of the modifiedtarget protein by OmpT1 increases the yield of proteins incorporatingnon-native amino acids in cell-free synthesis systems. The non-nativeamino acids used in the present invention typically comprise one or morechemically modified derivatives or analogues of amino acids, wherein thechemical structures have the formula NH3-(CR)—COOH, where R is not anyof the 20 canonical substituents defining the natural amino acids.Suitable non-native amino acid derivatives are commercially availablefrom vendors such as, e.g., Bachem Inc., (Torrance, Calif.); GenzymePharmaceuticals (Cambridge, Mass.); Senn Chemicals (Dielsdorf,Switzerland); Sigma-Aldrich (St. Louis, Mo.); Synthetec, Inc (Albany,Oreg.). Preferably, the non-native amino acids include but are notlimited to derivatives and/or analogs of glycine, tyrosine, glutamine,phenyalanine, serine, threonine, proline, tryptophan, leucine,methionine, lysine, alanine, arginine, asparagine, valine, isoleucine,aspartic acid, glutamic acid, cysteine, histidine, as well as beta-aminoacids and homologs, BOC-protected amino acids, and FMOC-protected aminoacids.

The generation of non-native amino acid derivatives, analogs andmimetics not already commercially available can be accomplished inseveral ways. For example, one way is to synthesize a non-native aminoacid of interest using organic chemistry methods known in the art, whileanother way is to utilize chemoenzymatic synthesis methods known in theart. See, e.g., Kamphuis et al., Ann. N. Y. Acad. Sci., 672:510-527,1992; Ager D J and Fotheringham I G, Curr. Opin. Drug Discov. Devel.,4:800-807, 2001; and Weiner et al., Chem. Soc. Rev., 39:1656-1691, 2010;Asymmetric Syntheses of Unnatural Amino Acids and HydroxyethylenePeptide Isosteres, Wieslaw M. Kazmierski, ed., PeptidomimeticsProtocols, Vol. 23, 1998; and Unnatural Amino Acids, Kumar G.Gadamasetti and Tamim Braish, ed., Process Chemistry in thePharmaceutical Industry, Vol. 2, 2008.

One skilled in the art will recognize that many procedures and protocolsare available for the synthesis of non-native amino acids, for example,as described in Wieslaw M. Kazmierski, ed., Peptidomimetics Protocols,Vol. 23, 1998; Wang L et al., Chemistry and Biology, 16:323-336, 2009;and Wang F, Robbins S, Guo J, Shen W and Schultz P G., PLoS One,5:e9354, 2010.

The non-native amino acids may include non-native L- and D-alpha aminoacids. L-alpha amino acids can be chemically synthesized by methodsknown in the art such as, but not limited to, hydrogen-mediatedreductive coupling via rhodium-catalyzed C—C bond formation ofhydrogenated conjugations of alkynes with ethyl iminoacetates (Kong etal., J. Am. Chem. Soc., 127:11269-11276, 2005). Alternatively,semisynthetic production by metabolic engineering can be utilized. Forexample, fermentation procedures can be used to synthesize non-nativeamino acids from E. coli harboring a re-engineered cysteine biosyntheticpathway. (see Maier T H, Nature, 21:422-427, 2003). Racemic mixtures ofalpha-amino acids can be produced using asymmetric Strecker syntheses(as described in Zuend et al., Nature, 461;968-970 (2009)) or usingtransaminase enzymes for large-scale synthesis (as found in Taylor etal., Trends Biotechnol., 16:412-419, 1998. Bicyclic tertiary alpha-aminoacids may be produced by alkylation of glycine-derived Schiff bases ornitroacetates with cyclic ether electrophiles, followed by acid-inducedring opening and cyclization in NH₄OH (see Strachan et al., J. Org.Chem., 71:9909-9911 (2006)).

The non-native amino acids may further comprise beta-amino acids, whichare remarkably stable to metabolism, exhibit slow microbial degradation,and are inherently stable to proteases and peptidases. An example of thesynthesis of beta amino acids is described in Tan C Y K and Weaver D F,Tetrahedron, 58:7449-7461, 2002.

In some instances, the non-native amino acids comprise chemicallymodified amino acids commonly used in solid phase peptide synthesis,including but not limited to, tert-butoxycarbonyl-(Boc) or(9H-fluoren-9-ylmethoxy)carbonyl (Fmoc)-protected amino acids. Forexample, Boc derivatives of leucine, methionine, threonine, tryptophanand proline can be produced by selective 3,3-dimethyldioxiraneside-chain oxidation, as described in Saladino et al., J. Org. Chem.,64:8468-8474, 1999. Fmoc derivatives of alpha-amino acids can besynthesized by alkylation of ethyl nitroacetate and transformation intoderivatives (see Fu et al., J. Org Chem., 66:7118-7124, 2001).

Non-native amino acids that can be used in the present invention mayinclude, but are not limited to, non-native analogues or derivatives ofthe 20 canonical amino acid substituents. One of skill in the art willappreciate that the synthesis of various non-native amino acids mayinvolve an array of chemical and chemo-enzymatic methods known in theart. In some embodiments, non-native amino acids may be synthesizedaccording to procedures known in the art specific to a particularderivative of each non-native amino acid. Sycheva et al., Microbiology,76:712-718, 2007 describes a procedure for synthesizing the non-nativeamino acids norvaline and norleucine. Diallylated proline derivativescan be produced by practical stereoselective synthesis (see Belvisi etal., Tetrahedron, 57:6463-6473, 2001). For example, tryptophanderivatives can be synthesized by ytterbium triflate catalyzedelectrophilic substitution of indo as described in Janczuk et al.,Tetrahedron Lett., 43:4271-4274, 2002, and synthesis of 5-aryltryptophan derivatives is detailed in Wang et al., Tetrahedron,58:3101-3110, 2002. Non-native serine analogs can be produced bybeta-fragmentation of primary alkoxyl radicals (see Boto et al., J. Org.Chem., 72:7260-7269, 2007). Alternatively, a procedure for phenylserinesynthesis is described in Koskinen et al., Tetrahedron Lett.,36:5619-5622, 1995. The procedure for the synthesis of L-phenylglycineis described in Cho et al., Biotechnol. Bioprocess. Eng., 11;299-305,2006. And a chemo-enzymatic method of synthesizingD-4-hydroxyphenylglycine is described in Yu et al., Folia Microbiol(Praha), 54:509-15; 2009. A non-limiting example of the production of2-naphthylalanine or Boc-protected 2-naphthylalanine is detailed in Boazet al., Org. Process Res. Dev., 9:472-478; 2005. Synthesis ofiodo-L-tyrosine and p-benzoyl-L-phenylalanine are described in Hino N,Nat. Protoc., 1:2957-2962, 2007.

Charged tRNA

In order to incorporate the non-native amino acids described herein intothe desired polypeptide, the nnAA described above need to be charged toisoaccepting sense or amber codon tRNAs. The tRNA charging reaction, asused herein, refers to the in vitro tRNA aminoacylation reaction inwhich desired isoaccepting sense codon or amber codon tRNAs areaminoacylated with their respective amino acid of interest. The tRNAcharging reaction comprises the charging reaction mixture, anisoaccepting sense tRNA, and as used in this invention, may includeeither natural or non-native amino acids. The tRNA charging reaction canoccur in situ in the same reaction as the cell-free translationreaction, or can occur in a separate reaction, where the charged tRNA isthen added to the cell-free translation reaction.

tRNA molecules to be used in the tRNA charging reaction can besynthesized from a synthetic DNA template for any tRNA of choicefollowing amplification by PCR in the presence of appropriate 5′ and 3′primers. The resulting double-stranded DNA template, containing aT7-promoter sequence, can then be transcribed in vitro using T7 RNApolymerase to produce the tRNA molecule, which is subsequently added tothe tRNA charging reaction.

The tRNA charging reaction can be any reaction that aminoacylates asense codon or amber codon tRNA molecule with a desired amino acidseparate from the protein synthesis reaction. This reaction can takeplace in an extract, an artificial reaction mixture, or a combination ofboth. Suitable tRNA aminoacylation reaction conditions are well known tothose of ordinary skill in the art. Typically, tRNA aminoacylation iscarried out in a physiological buffer with a pH value ranging from 6.5to 8.5, 0.5-10 mM high energy phosphate (such as ATP), 5-200 mM MgCl₂,20-200 mM KCl. Preferably, the reaction is conducted in the presence ofa reducing agent (such as 0-10 mM dithiothreitol). Where theaminoacyl-tRNA synthetase is exogenously added, the concentration of thesynthetase is typically 1-100 nM. One skilled in the art would readilyrecognize that these conditions can be varied to optimize tRNAaminoacylation, such as high specificity for the pre-selected aminoacids, high yields, and lowest cross-reactivity.

In other embodiments of the invention, isoaccepting or amber tRNAs arecharged by aminoacyl-tRNA synthetases. The tRNA charging reactions canutilize either the native aminoacyl-tRNA synthetase specific to theisoaccepting sense tRNAs to be charged, an engineered aminoacyl-tRNAsynthetase, or a “promiscuous” aminoacyl tRNA synthetase capable ofcharging a tRNA molecule with more than one type of amino acid.Promiscuous aminoacyl-tRNA synthetases may either themselves beengineered, or may include endogenously produced aminoacyl-tRNAsynthetases that are sometimes found in nature. Methods of chargingisoaccepting tRNAs with native and non-native amino acids usingaminoacyl-tRNA synthetases are described in WO2010/081110, the contentsof which are incorporated by reference herein.

Translation Systems

The above described charged isoaccepting sense or amber tRNAs are nowcombined with a translation system which can comprise a cell freeextract, cell lysate, or reconstituted translation system, along withthe nucleic acid template for synthesis of the desired polypeptide orprotein having non-native amino acids at preselected (defined)positions. The reaction mixture will further comprise monomers for themacromolecule to be synthesized, e.g. amino acids, nucleotides, etc.,and such co-factors, enzymes and other reagents that are necessary forthe synthesis, e.g. ribosomes, tRNA, polymerases, transcriptionalfactors, etc. In addition to the above components such as a cell-freeextract, nucleic acid template, and amino acids, materials specificallyrequired for protein synthesis may be added to the reaction. Thematerials include salts, folinic acid, cyclic AMP, inhibitors forprotein or nucleic acid degrading enzymes, inhibitors or regulators ofprotein synthesis, adjusters of oxidation/reduction potentials,non-denaturing surfactants, buffer components, spermine, spermidine,putrescine, etc. Various cell-free synthesis reaction systems are wellknown in the art. See, e.g., Kim, D. M. and Swartz, J. R. Biotechnol.Bioeng. 66:180-8 (1999); Kim, D. M. and Swartz, J. R. Biotechnol. Prog.16:385-90 (2000); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng.74:309-16 (2001); Swartz et al, Methods MoL Biol. 267:169-82 (2004);Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 85:122-29 (2004);Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng. 86:19-26 (2004);Yin, G. and Swartz, J. R., Biotechnol. Bioeng. 86:188-95 (2004); Jewett,M. C. and Swartz, J. R., Biotechnol. Bioeng. 87:465-72 (2004); Voloshin,A. M. and Swartz, J. R., Biotechnol. Bioeng. 91:516-21 (2005).Additional conditions for the cell-free synthesis of desiredpolypeptides are described in WO2010/081110, the contents of which areincorporated by reference herein in its entirety.

In some embodiments, a DNA template is used to drive in vitro proteinsynthesis, and RNA polymerase is added to the reaction mixture toprovide enhanced transcription of the DNA template. RNA polymerasessuitable for use herein include any RNA polymerase that functions in thebacteria from which the bacterial extract is derived. In otherembodiments, an RNA template is used to drive in vitro proteinsynthesis, and the components of the reaction mixture can be admixedtogether in any convenient order, but are preferably admixed in an orderwherein the RNA template is added last, thereby minimizing potentialdegradation of the RNA template by nucleases.

Cell Free Translation Systems

Cell-free protein synthesis can exploit the catalytic power of thecellular machinery. Obtaining maximum protein yields in vitro requiresadequate substrate supply, e.g. nucleoside triphosphates and aminoacids, a homeostatic environment, catalyst stability, and the removal oravoidance of inhibitory byproducts. The optimization of in vitrosynthetic reactions benefits from recreating the in vivo state of arapidly growing organism. In some embodiments of the invention,cell-free synthesis is therefore performed in a reaction where oxidativephosphorylation is activated, i.e. the CYTOMIM™ system. The CYTOMIM™system is defined by using a reaction condition in the absence ofpolyethylene glycol with optimized magnesium concentration. The CYTOMIM™system does not accumulate phosphate, which is known to inhibit proteinsynthesis, whereas conventional secondary energy sources result inphosphate accumulation. Various other features of the CYTOMIM™ systemare described in U.S. Pat. No. 7,338,789, the contents of which areincorporated by reference herein in its entirety.

The presence of an active oxidative phosphorylation pathway can bedemonstrated by the lack of a requirement for secondary energy sources,such as phosphoenolpyruvate, creatine phosphate, acetyl phosphate, orglycolytic intermediates such as glucose, glucose-6-phosphate, andpyruvate. The presence of an active oxidative phosphorylation pathwaycan also be determined by sensitivity of the pathway to inhibitors, suchas electron transport chain inhibitors. Examples of electron transportchain inhibitors include 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO),2,4-dinitrophenol, cyanide, azide, thenoyltrifluoroacetone, andcarbonyl-cyanide-m-chlorophenylhydrazone. Alternatively, in oneembodiment, the cell-free translation system does not comprise an activeoxidative phosphorylation pathway.

In vitro, or cell-free, protein synthesis offers several advantages overconventional in vivo protein expression methods. Cell-free systems candirect most, if not all, of the metabolic resources of the cell towardsthe exclusive production of one protein. Moreover, the lack of a cellwall and membrane components in vitro is advantageous since it allowsfor control of the synthesis environment. For example, tRNA levels canbe changed to reflect the codon usage of genes being expressed. Theredox potential, pH, or ionic strength can also be altered with greaterflexibility than with in vivo protein synthesis because concerns of cellgrowth or viability do not exist. Furthermore, direct recovery ofpurified, properly folded protein products can be easily achieved.

The productivity of cell-free systems has improved over 2-orders ofmagnitude in recent years, from about 5 μg/ml-hr to about 500 μg/ml-hr.Such improvements have made in vitro protein synthesis a practicaltechnique for laboratory-scale research and provides a platformtechnology for high-throughput protein expression. It further indicatesthe feasibility for using cell-free technologies as an alternative meansto in vivo large-scale, commercial production of proteinpharmaceuticals.

Generating a Lysate

The present invention utilizes a cell lysate for in vitro translation ofa target protein. For convenience, the organism used as a source for thelysate may be referred to as the source organism or host cell. Hostcells may be bacteria, yeast, mammalian or plant cells, or any othertype of cell capable of protein synthesis. A lysate comprises componentsthat are capable of translating messenger ribonucleic acid (mRNA)encoding a desired protein, and optionally comprises components that arecapable of transcribing DNA encoding a desired protein. Such componentsinclude, for example, DNA-directed RNA polymerase (RNA polymerase), anytranscription activators that are required for initiation oftranscription of DNA encoding the desired protein, transfer ribonucleicacids (tRNAs), aminoacyl-tRNA synthetases, 70S ribosomes,N¹⁰-formyltetrahydrofolate, formylmethionine-tRNAf^(met) synthetase,peptidyl transferase, initiation factors such as IF-1, IF-2, and IF-3,elongation factors such as EF-Tu, EF-Ts, and EF-G, release factors suchas RF-1, RF-2, and RF-3, and the like.

An embodiment uses a bacterial cell from which a lysate is derived. Abacterial lysate derived from any strain of bacteria can be used in themethods of the invention. The bacterial lysate can be obtained asfollows. The bacteria of choice are grown up overnight in any of anumber of growth media and under growth conditions that are well knownin the art and easily optimized by a practitioner for growth of theparticular bacteria. For example, a natural environment for synthesisutilizes cell lysates derived from bacterial cells grown in mediumcontaining glucose and phosphate, where the glucose is present at aconcentration of at least about 0.25% (weight/volume), more usually atleast about 1%; and usually not more than about 4%, more usually notmore than about 2%. An example of such media is 2YTPG medium, howeverone of skill in the art will appreciate that many culture media can beadapted for this purpose, as there are many published media suitable forthe growth of bacteria such as E. coli, using both defined and undefinedsources of nutrients. Cells that have been harvested overnight can belysed by suspending the cell pellet in a suitable cell suspensionbuffer, and disrupting the suspended cells by sonication, breaking thesuspended cells in a French press, continuous flow high pressurehomogenization, or any other method known in the art useful forefficient cell lysis The cell lysate is then centrifuged or filtered toremove large DNA fragments.

EXAMPLES Example 1

This example describes the modification of RF1 protein to introduceamino acid sequences that are cleavable by OmpT1 protease.

Methods:

Construction of a Template to Introduce OMPT1 Cleavage Sites into RF1

A PCR based strategy was used to introduce desired nucleotide sequencechanges into nucleic acid templates that encode the modified RF1proteins described herein. The PCR reaction was carried out usingPhusion Hot Start Flex 2X Master Mix (NEB) according to the protocolssuggested by the manufacturer. Generally a two-step overlapping PCR wascarried out to introduce OmpT cleavage mutations into the RF1 encodinggene, as described in more detail below. PCR generated DNA templateswere purified using QIAquick PCR purification kit (QIAGEN) forapplication in cell-free expression. After PCR purification the variantswere sequenced by Mclab (South San Francisco, Calif.) to confirm thepresence of the expected mutations.

Preparation of GamS Protein to Prevent Degradation of DNA Templates inCell-Free Reactions

The efficiency of transcription from a DNA template can be decreased dueto degradation of the template by endogenous bacterial exonucleasespresent in cell-free extracts. The short form of λ phage Gam protein(GamS) is known to protect DNA templates from degradation by inhibitingthe activity of RecBCD (Exonuclease V) (see Sitararman, K., Esposito,D., Klarmann, G., Grice, S. F. L., Hartley, J. L. and Chatterjee, D. K.2004, A Novel Cell-free Protein Synthesis System. J Biotechnol. 110:257-263. Therefore, GamS protein was used in this example to stabilizePCR templates during cell-free transcription reactions. To producerecombinant GamS protein, the GamS gene was amplified to include aC-terminal poly-histidine tag, GGSHHHHHH (SEQ ID NO:50), by primers,5′-ATATATCATATGAACGCTTATTACATTCAGGATCGTCTTGAG-3′ (SEQ ID NO:51), and5′-ATATATGTCGACTTAATGATGATGATGATGATGAGAACCCCCTACCTCTGAATCAATATCAACCTGGTGGTG-3′(SEQ ID NO:52) using pKD46 (Datsenko, K. A. and Wanner, B. 2000,One-step Inactivation of Chromosomal Genes in Escherichia coli K-12Using PCR Products. Proc. Natl. Acad. Sci. USA 97: 6640-6645) astemplate. The GamS gene was subcloned into the cell-free expressionplasmid pYD317 at NdeI/SalI restriction sites. GamS was expressed invitro and purified by Immobilized Metal Affinity Chromatography (IMAC)with purity higher than 90% (data not shown). GamS protein was stored at−70° C. before application in 100 mM Tris-Acetate buffer (pH 8.2), whichalso contained 160 mM potassium acetate, 200 mM sodium chloride and 10%sucrose.

Bacterial Strains and Plasmids

SBJY001 is an E. coli K12 derivative optimized for open cell freeprotein production. SBJY001 was transformed with the plasmid pKD46 (ColiGenetic Stock Center) which contains the phage λ Red recombinase genesunder an inducible arabinose promoter.

Construction of SBHS002

SBHS002 is an ompT deletion E. coli strain created using P1transduction. P1 lysate was made from JW0554-1 (CGSC#8680), a Keiocollection strain containing the ompT::Kan^(R) mutation flanked by FRTsites. The JW0554-1 P1 lysate was then used to introduce the mutationinto SBJY001 by P1 transduction. Colonies were grown on LB with 30 μg/mlof kanamycin to select for kanamycin resistance. SBJY001ompT::Kan^(R)was transformed with the 708-FLPe Cm^(R) expression plasmid (GeneBridges). FLP synthesis was induced and colonies were screened for theloss of kanamycin resistance. Kanamycin resistant colonies weresequenced to confirm the deletion of ompT. The ompT deleted strain isreferred to as SBHS002.

Cloning and Expression of N-Terminal His-Tagged RF1

RF1 was amplified from E. coli strain A19 genomic DNA using primers5His-RF1:CATATGCATCACCATCACCATCACGGTGGTGGCTCTAAGCCTTCTATCGTTGCCAAACTGGAAGCC (SEQID NO:138) and 3RF1: GTCGACTTATTCCTGCTCGGACAACGCCGCCAG (SEQ ID NO:139)that introduced an N-terminal His-Tag and NdeI/SalI restriction sites.The insert was ligated into the expression vector pYD317 and confirmedby sequencing. RF1 was expressed in a 25 mL cell free reaction using theplasmid, purified by IMAC, and buffer exchanged into PBS.

Cleavage of Recombinant RF1 Variants Using Cell-Free Extracts

To test if the modified RF1 proteins described herein were cleavable byOmpT, recombinant RF1 variants were incubated with cell extracts with orwithout OmpT. SBJY001 cells, which have intact ompT protease on theouter membrane, and SBHS002, in which ompT was deleted, were grown up in5 mL of LB overnight at 37° C. 50 μL of each culture was spun down at8,000 rpm for 2 minutes and washed twice with 10 mM Tris, 20 mM ammoniumchloride and 10 mM magnesium chloride. The cell pellets were thenresuspended in 50 uL of the buffer and 10 μg of purified recombinant E.coli RF1 protein was added. The samples were incubated at 37° C. Thesamples were spun down at 8,000 rpm for 2 minutes. The supernatantcontaining the RF1 protein was removed and run on a SDS-PAGE gel.

Cell-Free Expression of RF1 Variants

The cell free transcription/translation reactions were carried out in avolume of 60 μl at 30° C. in 24 deep well plates (Cat. No.95040470,Thermo Scientific) for 4.5 hours. The PCR template concentration for RF1variant expression was 10 μg/ml. The reaction composition also included8 mM magnesium glutamate, 130 mM potassium glutamate, 35 mM sodiumpyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP and CMP, 4 mM sodiumoxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate,1 mM tyrosine, 2 mM of each 19 other amino acids, 100 nM T7 RNApolymerase, 30% (V/V) S30 cell-extract. To facilitate disulfideformation, S30 cell-extract was treated with 50 μM IAM at roomtemperature for 30 min before cell-free reaction. A mixture of 2 mMoxidized glutathione (GSSG) and 1 mM reduced glutathione (GSH) was alsoadded with 4.3 μM E. coli disulfide isomerase DsbC. To analyze cell-freeexpressed RF1 variants with SDS-PAGE and autoradiogram, reactions wereperformed in the presence of trace amounts of [¹⁴C]-leucine (300μCi/mole; GE Life Sciences, N.J.). The RF1 variants were expressed ineither an OmpT-positive (OmpT+) cell-extract, which was prepared frombacterial strain SBJY001, or an OmpT-negative (OmpT−) cell-extract,which was prepared from bacterial strain SBHS002 (SBJY001ΔompT). Tostabilize PCR templates in the cell-free reaction, 1.4 μM GamS proteinwas also added to inhibit the activity of RecBCD.

SDS-PAGE and Autoradiography

In order to determine if the modified RF1 proteins were cleavable byOmpT, the RF1 proteins translated in the cell free reactions describedabove were analyzed by SDS-PAGE and autoradiography. The cell-freereaction samples labeled with ¹⁴C were centrifuged at the maximum speedin a bench top centrifuge and 4 μL of supernatant was mixed withInvitrogen SDS-PAGE sample loading buffer and water. The samples wereloaded on 4˜12% Bis-Tris SDS-PAGE gels and run with MES running bufferfor about 45 minutes. Then the gels were dried and exposed to phosphorscreen (63-0034-86, GE healthcare, USA) overnight, and then scannedusing Storm 460 (GE healthcare, USA).

Introduction of OmpT Cleavage Sites into RF1

To identify potential OmpT cleavage sites in the RF1 sequence, singleArg or Lys mutations were introduced into different loop regions of RF1beside an existing Arg or Lys. The loop regions were predicted based onsequence alignment of prokaryotic class I release factors (see Graille,M., Heurgue-Hamard, V., Champ, S., Mora, L., Scrima, N., Ulryck, N.,Tilbeurgh, H. and Buckingham, R. H. 2005, Molecular Basis for BacterialClass I Release Factor Methylation by PrmC. Molecular Cell. 20:917-927.) To generate the desired variants, the mutation sequences weredesigned in the middle of the forward and reverse oligo primers (Table1). In the first step PCR, the 5′-fragment was amplified by PCR using5chiT2PT7,5′-GCGTACTAGCGTACCACGTGGCTGGTGGCCGATTCATTAATGCAGCTGGCACGACAGG-3′ (SEQ IDNO:53), and the reverse primers (Table 1). The 5′fragment included T7promoter, the constant region of the N-terminal sequence and themutation site. The 3′-fragment was also amplified in the first stepusing forward primers (Table 1) and 3chiT2TT7,5′-GCGTACTAGCGTACCACGTGGCTGGTGGCGGTGAGTTTTCTCCTTCATTACAGAAACGGC-3’ (SEQID NO:54). The 3′ fragment included the mutation site, the constantC-terminal region and T7 terminator sequences. In the second step PCR,the 5′-fragment and 3′-fragment DNA were assembled by overlapping PCRusing a single primer 5chiT2,5′-GCGTACTAGCGTACCACGTGGCTGGTGG-3′ (SEQ IDNO:55).

First, 9 single mutations, M74R, E76R, E84R, A85R, E87R, E108R, T293R,N296K and S304R, were introduced in the loop regions beside an existingArg or Lys (Table 2). These RF1 variants and WT RF1 were expressed usingPCR templates in cell-free extracts with or without OmpT. The RF1proteins were analyzed by SDS-PAGE and autoradiography. When expressedin an OmpT negative cell-extract, all 10 RF1 variants showed completefull-length protein on SDS-PAGE. However, when expressed incell-extracts containing OmpT, variant N296R was partially digestedwhile the other variants migrated as expected for full-length,undigested proteins. N296 is located in the switch loop region of RF1,which is flexible and easy to access by protease digestion.

In the first round screening, N296K was selected for partial digestionby OmpT in cell-extract. In the second round screening, PCR templates ofdouble mutant and triple mutant variants were generated using the samemethod as described above with primers listed in Table 1. Six doublemutants, N296K/L297V, N296K/L297K, N296K/L297R, N296R/L297V,N296R/L297K, N296R/L297R, and 2 triple mutants, N296K/L297R/L298K andN296K/L297R/L298R (Table 2), were tested in cell-free expression with orwithout OmpT. All eight double and triple RF1 mutant variants werecleaved by OmpT1 (data not shown). Among these variants,N296K/L297R/L298R was most sensitive to digestion by OmpT.

TABLE 1Forward and reverse primer sequences used in OmpT cleavage site screeningSEQ ID RF1 Variants NO: Forward WT GCTCGATGATCCTGAAATGCGTGAGATGGCGCAGG56 M74R GCTCGATGATCCTGAACGCCGTGAGATGGCGCAGG 57 E76KCGATGATCCTGAAATGCGTAAGATGGCGCAGGATGAAC 58 E84KCAGGATGAACTGCGCAAAGCTAAAGAAAAAAGCGAGCAAC 59 A85RCAGGATGAACTGCGCGAACGTAAAGAAAAAAGCGAGCAAC 60 E87RGGATGAACTGCGCGAAGCTAAACGTAAAAGCGAGCAACTGGAAC 61 E108RGCCAAAAGATCCTGATGACCGTCGTAACGCCTTCCTCG 62 T293RCAACAGGCCGAAGCGTCTCGCCGTCGTAACCTGC 63 N296KGCGTCTACCCGTCGTAAACTGCTGGGGAGTGGCG 64 S304KGGGAGTGGCGATCGCAAGGACCGTAACCGTACTTAC 65 N296K/L297VCGAAGCGTCTACCCGTCGTAAAGTTCTGGGGAGTGGCGATCGCAGC 66 N296K/L297KCGAAGCGTCTACCCGTCGTAAAAAGCTGGGGAGTGGCGATCGCAGC 67 N296K/L297RCGAAGCGTCTACCCGTCGTAAACGTCTGGGGAGTGGCGATCGCAGC 68 N296R/L297VCGAAGCGTCTACCCGTCGTCGCGTTCTGGGGAGTGGCGATCGCAGC 69 N296R/L297KCGAAGCGTCTACCCGTCGTCGCAAGCTGGGGAGTGGCGATCGCAGC 70 N296R/L297RCGAAGCGTCTACCCGTCGTCGCCGTCTGGGGAGTGGCGATCGCAGC 71 N296K/L297R/L298KCAGGCCGAAGCGTCTACCCGTCGTAAACGTAAGGGGAGTGGCGATCGCAGCGACC 72N296K/L297R/L298RCAGGCCGAAGCGTCTACCCGTCGTAAACGTCGCGGGAGTGGCGATCGCAGCGACC 73 Reverse WTCCTGCGCCATCTCACGCATTTCAGGATCATCGAGC 74 M74RCCTGCGCCATCTCACGGCGTTCAGGATCATCGAGC 75 E76KGTTCATCCTGCGCCATCTTACGCATTTCAGGATCATCG 76 E84KGTTGCTCGCTTTTTTCTTTAGCTTTGCGCAGTTCATCCTG 77 A85RGTTGCTCGCTTTTTTCTTTACGTTCGCGCAGTTCATCCTG 78 E87RGTTCCAGTTGCTCGCTTTTACGTTTAGCTTCGCGCAGTTCATCC 79 E108RCGAGGAAGGCGTTACGACGGTCATCAGGATCTTTTGGC 80 T293RGCAGGTTACGACGGCGAGACGCTTCGGCCTGTTG 81 N296KCGCCACTCCCCAGCAGTTTACGACGGGTAGACGC 82 S304KGTAAGTACGGTTACGGTCCTTGCGATCGCCACTCCC 83 N296K/L297VGCTGCGATCGCCACTCCCCAGAACTTTACGACGGGTAGACGCTTCG 84 N296K/L297KGCTGCGATCGCCACTCCCCAGCTTTTTACGACGGGTAGACGCTTCG 85 N296K/L297RGCTGCGATCGCCACTCCCCAGACGTTTACGACGGGTAGACGCTTCG 86 N296R/L297VGCTGCGATCGCCACTCCCCAGAACGCGACGACGGGTAGACGCTTCG 87 N296R/L297KGCTGCGATCGCCACTCCCCAGCTTGCGACGACGGGTAGACGCTTCG 88 N296R/L297RGCTGCGATCGCCACTCCCCAGACGGCGACGACGGGTAGACGCTTCG 89 N296K/L297R/L298KGGTCGCTGCGATCGCCACTCCCCTTACGTTTACGACGGGTAGACGCTTCGGCCTG 90N296K/L297R/L298RGGTCGCTGCGATCGCCACTCCCGCGACGTTTACGACGGGTAGACGCTTCGGCCTG 91 *Mutationsequences are underlined.

TABLE 2 Single, double and triple substitution variants of RF1 No.Single Substitution Variants A-1 M74R A-2 E76K A-3 E84K A-4 A85R A-5E87R A-6 E108R A-7 T293R A-8 N296K A-9 S304K Double SubstitutionVariants A-11 N296K/L297V A-12 N296K/L297K A-13 N296K/L297R A-14N296R/L297V A-15 N296R/L297K A-16 N296R/L297R Triple SubstitutionVariants A-17 N296K/L297R/L298K A-18 N296K/L297R/L298RInserting OmpT Cleavage Peptides into the Switch Loop Region of RF1

In addition to introducing the amino acid mutations described above, RF1was also modified to replace wild-type sequences in the switch loopregion with known OmpT protease-susceptible peptide sequences (seeHwang, B., Varadarajan, N., Li, H., Rodriguez, S., Iverson, B. L. andGeorgiou, G. 2007, Substrate Specificity of the Escherichia coli OuterMembrane Protease OmpP. J. Bacteriol. 189: 522-530; McCarter, J. D.,Stephens, D., Shoemaker, K., Rosenberg, S., Kirsch, J. F. and Georgiou,G. 2004, Substrate Specificity of the Escherichia coli Outer MembraneProtease OmpT. J. Bacteriol. 186: 5919-5925). 22 RF1 variants wereconstructed, and are listed in Table 3. Variant Nos. 1 to 14 containedARRG (SEQ ID NO:47) for OmpT digestion. Variant No. 15 contained ARRinstead of ARRG (SEQ ID NO:47) since it is at the end of switch loop.Variant No. 16 contained a single mutation N296R. Variant Nos. 17, 18and 19 contained an OmpT cleavage peptide WLAARRGRG (SEQ ID NO:48).Variant Nos. 20, 21 and 22 contained another OmpT cleavage peptideWGGRWARKKGTI (SEQ ID NO:49).

The mutation sequences were designed in the forward and reverse oligoprimers listed in Table 4. In the first step PCR, the 5′-fragment wasamplified by PCR using the primer 5chiT2PT7 and the reverse primers(Table 4). The 5′fragment included T7 promoter, the constant region ofthe N-terminal sequence and the mutation site. The 3′-fragment was alsoamplified in the first step using forward primers (Table 3) and3chiT2TT7. The 3′ fragment included the mutation site, the constantC-terminal region and T7 terminator sequences. In the second step PCR,the 5′-fragment and 3′-fragment DNA were assembled by overlapping PCRusing a single primer 5chiT2 as described above.

22 RF1 variants (Table 3) were expressed using PCR templates incell-extracts with or without OmpT. Among the ARRG (SEQ ID NO:47)insertion variants, No.9 showed the highest sensitivity to OmpTdigestion. Variant Nos. 17, 18 and 19, which contained the OmpT cleavagepeptide WLAARRGRG (SEQ ID NO:48), were partially digested by OmpT.However, they were much less sensitive than variant Nos. 20, 21 and 22,which contained the OmpT cleavage peptide WGGRWARKKGTI (SEQ ID NO:49).Variant Nos. 9, 20, 21 and 22 were selected as the most sensitive RF1variants to OmpT digestion in these 22 constructs.

TABLE 3 OmpT cleavage peptide sequences inserted in the switch loop of RF1 SEQ  ID No, Peptide** NO:  *0QQAEASTRRNLLGSGDRS  4   1 QARRGSTRRNLLGSGDRS 26   2 QQARRGTRRNLLGSGDRS27   3 QQAARRGRRNLLGSGDRS 28   4 QQAEARRGRNLLGSGDRS 29   5QQAEAARRGNLLGSGDRS 30   6 QQAEASARRGLLGSGDRS 31   7 QQAEASTARRGLGSGDRS32   8 QQAEASTRARRGGSGDRS 33   9 QQAEASTRRARRGSGDRS 34  10QQAEASTRRNARRGGDRS 35  11 QQAEASTRRNLARRGDRS 36  12 QQAEASTRRNLLARRGRS37  13 QQAEASTRRNLLGARRGS 38  14 QQAEASTRRNLLGSARRG 39  15QQAEASTRRNLLGSGARR 40  16 QQAEASTRRRLLGSGDRS  6  17 QQAWLAARRGRGGSGDRS41  18 QQAEWLAARRGRGSGDRS 42  19 QQAEAWLAARRGRGGDRS 43  20QQWGGRWARKKGTIGDRS 44  21 QQAWGGRWARKKGTIDRS 45  22 QQAEWGGRWARKKGTIRS46 *WT switch loop peptide sequence (Q287 to S304) **The inserted aminoacid or peptide sequences are underlined

TABLE 4Forward and reverse primer sequences for OmpT cleavage peptide insertionSEQ ID No. NO: Forward  0GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACT  92TACAACTTCCCG  1CGCCGTGGTTCTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 93 CCCG  2GCACGCCGTGGTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 94 CCCG  3GCCGCACGCCGTGGTCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 95 CCCG  4GCCGAAGCACGCCGTGGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 96 CCCG  5GCCGAAGCGGCACGCCGTGGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACT 97 TCCCG  6GCCGAAGCGTCTGCACGCCGTGGTCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 98 CCCG  7GCCGAAGCGTCTACCGCACGCCGTGGTCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT 99 CCCG  8GCCGAAGCGTCTACCCGTGCACGCCGTGGTGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT100 CCCG  9GCCGAAGCGTCTACCCGTCGTGCACGCCGTGGTAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT101 CCCG 10GCCGAAGCGTCTACCCGTCGTAACGCACGCCGTGGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT102 CCCG 11GCCGAAGCGTCTACCCGTCGTAACCTGGCACGCCGTGGTGATCGCAGCGACCGTAACCGTACTTACAACTT103 CCCG 12GCCGAAGCGTCTACCCGTCGTAACCTGCTGGCACGCCGTGGTCGCAGCGACCGTAACCGTACTTACAACTTC104 CCG 13GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGGCACGCCGTGGTAGCGACCGTAACCGTACTTACAACTT105 CCCG 14GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGCACGCCGTGGTGACCGTAACCGTACTTACAACTT106 CCCG 15GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGGCGCACGCCGTGACCGTAACCGTACTTACAACTT107 CCCG 16GCCGAAGCGTCTACCCGTCGTCGTCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT108 CCCG 17GCCTGGCTGGCAGCGCGTCGCGGTCGTGGCGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACT109 TCCCG 18GCCGAATGGCTGGCAGCGCGTCGCGGTCGTGGCAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACT110 TCCCG 19GCCGAAGCGTGGCTGGCAGCGCGTCGCGGTCGTGGCGGCGATCGCAGCGACCGTAACCGTACTTACAACT111 TCCCG 20TGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTGGCGATCGCAGCGACCGTAACCGTACTTACAACTT112 CCCG 21GCCTGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTGATCGCAGCGACCGTAACCGTACTTACAACTT113 CCCG 22GCCGAATGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTCGCAGCGACCGTAACCGTACTTACAACTT114 CCCG Reverse  0GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC115 AGC  1GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTAGAACCACGGCGTGCTTGGCGTTTTGCCATTTCAG116 CAGC  2GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTACCACGGCGTGCCTGTTGGCGTTTTGCCATTTCAGC117 AGC  3GCTGCGATCGCCACTCCCCAGCAGGTTACGACGACCACGGCGTGCGGCCTGTTGGCGTTTTGCCATTTCAGC118 AGC  4GCTGCGATCGCCACTCCCCAGCAGGTTACGACCACGGCGTGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC119 AGC  5GCTGCGATCGCCACTCCCCAGCAGGTTACCACGGCGTGCCGCTTCGGCCTGTTGGCGTTTTGCC 120ATTTCAGCAGC  6GCTGCGATCGCCACTCCCCAGCAGACCACGGCGTGCAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC121 AGC  7GCTGCGATCGCCACTCCCCAGACCACGGCGTGCGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC122 AGC  8GCTGCGATCGCCACTCCCACCACGGCGTGCACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC123 AGC  9GCTGCGATCGCCACTACCACGGCGTGCACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG124 CAGC 10GCTGCGATCGCCACCACGGCGTGCGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG125 CAGC 11GCTGCGATCACCACGGCGTGCCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG126 CAGC 12GCTGCGACCACGGCGTGCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG127 CAGC 13GCTACCACGGCGTGCCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG128 CAGC 14ACCACGGCGTGCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC129 AGC 15ACGGCGTGCGCCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG130 CAGC 16GCTGCGATCGCCACTCCCCAGCAGACGACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAG131 CAGC 17GCTGCGATCGCCACTCCCGCCACGACCGCGACGCGCTGCCAGCCAGGCCTGTTGGCGTTTTGCCATTTCAGC132 AGC 18GCTGCGATCGCCACTGCCACGACCGCGACGCGCTGCCAGCCATTCGGCCTGTTGGCGTTTTGCCATTTCAGC133 AGC 19GCTGCGATCGCCGCCACGACCGCGACGCGCTGCCAGCCACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGC134 AGC 20GCTGCGATCGCCAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCACTGTTGGCGTTTTGCCATTTCAGC135 AGC 21GCTGCGATCAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCAGGCCTGTTGGCGTTTTGCCATTTCAGC136 AGC 22GCTGCGAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCATTCGGCCTGTTGGCGTTTTGCCATTTCAGC137 AGC *Mutation sequences are underlined.

Example 2

Having demonstrated that RF1 was successfully modified to be cleavableby OmpT1 protease, this example describes the construction ofrecombinant bacterial strains that express both modified RF1 and intactOmpT1. The purpose of this example is to show that RF1 variantsexpressed by the recombinant strains are cleaved in cell-free extractsfrom the recombinant strains that express OmpT1.

Oligonucleotide-Mediated Allelic Replacement

In order to generate bacterial strains that express the modified RF1proteins described herein, oligonucleotide-mediated allelic replacement(OMAR) was used to insert the RF1 mutations into the bacterial genome.The OMAR protocol was adapted from a previously reported protocol (Wangand Church, Methods in Enzymology, 2011, 498, 409-426). Briefly, SBJY001containing the pKD46 plasmid were grown in 3 ml LB and 50 μg/mLampicillin at 30° C. to OD₆₀₀ 0.3. The cells were then induced with 1 mML-arabinose at 37° C. for 45 min. The cell pellet was washed 2× withcold 10% glycerol and resuspended in 30 μL cold 10% glycerol. 5 μM ofeach oligo was added to the resuspended cells. Synthetic oligos(Integrated DNA Technologies) were 90 base pairs long and designed toanneal to the lagging strand during DNA replication (see Table 5). Thecells were electroporated at 1800V for 5 ms in a 1 mm cuvette. They werethen recovered in 3 mL LB and 50 μg/ml Amp. This process was repeatedfor 13 cycles. Cells were diluted and plated on LB agar plates and grownat 37° C. overnight.

MAMA PCR to Identify Bacterial Strains with the Desired Mutations

Bacterial colonies were screened using an adaptation of MismatchAmplification Mutation Assay (MAMA) PCR to identify strains with thedesired mutations in RF1 (Cha et. Al, PCR Methods and Applications,1992, 2, 14-20). Briefly, a universal 5′ primer was used in conjunctionwith a 3′ primer that was specific for each mutation to differentiatebetween a mutant and a WT colony (see Table 5). The oligos were orderedfrom Eurofins MWG Operon. Platinum® Blue PCR Supermix (Invitrogen) wasused to run the MAMA PCR. The PCR was run at 95° C. 3 min, 30× (95° C.15 sec, 58° C. 20 sec, 72° C. 1 min) and 72° C. 5 min. The PCR productswere run on a 96 well E-gel (Invitrogen) to visualize any bands.

Extract Preparation and Western Blot

Strains SBHS015, SBHS016 and SBHS017, that were engineered to containmodified RF1 variants, were grown up in 500 mL TB at 37° C. shakingovernight in Tunair shake flasks. The cells were pelleted at 6000×g for15 minutes. The cell pellet was washed 2× with 6 mL S30 Buffer (10 mMTris, 14 mM magnesium acetate and 60 mM potassium acetate): 1 g cellpellet. The cells were then resuspended in 2 mL S30 Buffer: 1 g cellpellet. The resuspended cells were lysed using a homogenizer. Theextract was then clarified 2× at 15,000×g for 30 minutes. The extractwas activated for 1, 2 or 3 hrs in a 30° C. water bath. An anti-RF1antibody was made by inoculating rabbits with purified recombinant E.coli RF1 protein that was then purified using an affinity matrix (YenZymAntibodies LLC). The specificity of the antibody was confirmed usingELISAs and Western Blots of the recombinant protein. The cell pellet,lysate and extract samples were run on a SDS-PAGE gel and transferred toa PVDF membrane using the iBlot® system (Invitrogen). The primaryanti-RF1 antibody was used followed by a secondary anti-rabbitalkaline-phosphatase conjugated antibody (Invitrogen). The bands werevisualized using an alkaline-phosphatase chromogenic substrate solutioncontaining 5-bromo-4-chloro-3-indolyl-1-phosphate and nitrobluetetrazolium (Invitrogen).

TABLE 5 Sequences of oligonucleotides for OMAR and MAMA PCR. SED IDOligo name Oligo Sequence (5′ to 3′) NO: 1opRF1 KRGGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCaAGac 140 (OMAR)GcTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCC 1opRF1 KRRGGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCacgacG 141 (OMAR)cTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCC 1opRF1 KRKGGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCottacG 142 (OMAR)cTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCC 3KR op-PCRGCG ATC CCC TGA ACC AAG ACG C 143 (MAMA) 3 KRR op- CGATCcCCtgaaCCacgacGc144 PCR (MAMA) 3 KRK op- TGCGATCcCCtgaaCCcttacGc 145 PCR (MAMA)5 RF1 op- CGTGACGGGGATAACGAACGCC 146 PCR (MAMA)

Three recombinant bacterial strains were identified that successfullyincorporated mutations of RF1 into their genomes. Strain SBHS015contains the N296K/L297R double mutation variant of RF1, referred to asvariant A13. Strain SBHS016 contains the N296K/L297R/L298R triplemutation variant of RF1, referred to as variant A18. Strain SBHS017contains the N296K/L297R/L298K triple mutation variant of RF1, referredto as variant A17. All three strains also express intact OmpT1, as theyare derived from the parent strain SBJY001. When the recombinant strainswere lysed and incubated for various time periods (0, 1, 2 and 3 hours),the modified RF1 protein variants were efficiently cleaved at all timepoints tested by the cell-free extract, as determined by Western blotanalysis (data not shown). In contrast, cleavage of unmodified,wild-type RF1 by strain SBJY001 was not detected over the same timeperiods, indicating that wild-type RF1 is not efficiently cleaved incell-free extracts containing intact OmpT1.

This example demonstrates that recombinant bacterial strains wereengineered to express modified RF1 variants, and that the RF1 variantswere cleaved by cell-free extracts from OmpT1 positive strains.

EXAMPLE 3

Example 3 demonstrates that intact RF1 proteins modified to includeOmpT1 cleavage sites in the switch loop region have wild-type RF1function.

Functional Test of Recombinant RF1 Variants

In order to test the function of the recombinant RF1 variants describedherein, the ability of the RF1 variants to terminate translation at anamber codon was determined. An Fc protein with a TAG mutation wasexpressed in the presence of 500 nM of purified recombinant E. coliwild-type or mutant RF1 and 2 μM non-natural amino acid in SBHS002extract (an OmpT deleted strain). The 60 μL cell free reactions were runat 30° C. for 5 hrs in the presence of ¹⁴C-Leu. The final reactions werecentrifuged to obtain the soluble fraction, run on a reducing SDS-PAGEgel, transferred to a PVDF membrane (Invitrogen), and exposed to aphosphoscreen overnight. The phosphoscreen was visualized using a StormImager and ImageQuant was used to determine the relative bandintensities. Relative activities of mutants were determined by comparingthe amount of truncated Fc protein to the negative control (no exogenousRF1 added) and the positive control (WT RF1 added). The percenttruncated protein was determined using the equation: (truncated proteincounts/total protein counts)×100%. Relative RF1 activity was determinedusing the equation: [(variant truncated protein−negative controltruncated protein)/(WT RF1 truncated protein−negative control truncatedprotein)]×100%.

As shown in FIG. 1, RF1 variants described herein have RF1 activity, asdemonstrated by truncation of translation when incorporating anon-native amino acid (pAzF) at an amber codon introduced at positionS378 of the Fc protein. In particular, the RF1 variant A13 (havingN296K/L297R substitutions) possessed similar activity levels aswild-type RF1.

This example demonstrates that intact RF1 proteins modified to includeOmpT cleavage sites in the switch loop region have functional RF1activity (e.g., reduced amber suppression).

Example 4

Example 4 demonstrates increased incorporation of non-natural aminoacids into the IgG heavy chain of Herceptin protein using cell freeextracts comprising RF1 variants having OmpT1 cleavage sites in theswitch loop region. The cell free extracts are from the bacterialstrains described in Example 2.

Methods

Site Directed Mutagenesis of Herceptin Heavy Chain

To introduce a nnAA into the heavy chain of Herceptin, the DNA templateencoding Herceptin was mutated to introduce amber codons at differentpositions of the coding sequence. Site directed mutagenesis wasperformed using a pYD plasmid containing the coding region ofHerceptin6×His at the C-terminus as the DNA template and syntheticoligonucleotides (Operon) containing amber codons in both sense andantisense directions (Table 6). Oligonucleotides of each mutation weremixed with the DNA template and Phusion® polymerase (Thermo, Cat# F531s)to a final volume of 20 μL. The final concentration of each componentwas 0.16 μM of each oligonucleotide, 0.5 ng/μL template DNA, 0.02 U/μLPhusion® polymerase in HF buffer (Thermo) containing 1.5 mM MgCl₂ and200 μM dNTP. Mixture was incubated at 98° C. 5 m, 18 PCR cycles (98° C.30 s, 55° C. 1 m, 72° C. 4 m), 10 m at 72° C. and stored at 4° C. for upto 16 h. DpnI (NEB) was added to the mixture to final concentration of0.6 U/μL and incubated for 37° C. 1 h. 5 μL of each mixture wastransformed into 50 μL of Chemically Competent E. coli cells accordingto manufactures procedure (Invitrogen, MultiShot™ 96-Well Plate TOP10).Transformed cells were recovered in 200 μL SOC (Invitrogen) 37° C. 1 hand plated onto Luria-Bertani (LB) agar supplemented with 50 μg/mLkanamycin (Teknova). After 24 h at 37° C., colonies were picked usingQpix2 (Genetix) into 200 μL LB with 7.5% glycerol and 50 μg/mLkanamycin, and grown at 37° C. for 24 h, 20 μL of culture was used forrolling circle amplification and sequenced by primer extension using T7(5′-TAATACGACTCACTATAGG-3′; SEQ ID NO:147) and T7 term(5′-GCTAGTTATTGCTCAGCG-3′; SEQ ID NO:148) primers (Sequetech). Sequencewas analyzed by Sequencher (Gene Codes).

TABLE 6 Primers for introducing amber codons at the indicated positions of Herceptin. Sense Oligo Antisense Oligo Variant (SEQ ID NO:)(SEQ ID NO:) SP-00067_V422 CGTTGGCAGCAGGGTAATTAGTTCATAACGCTGCAGCTGAACTAA CAGCTGCAGCGTTATG TTACCCTGCTGCCAACG (149) (150)SP-00127_S415 GCAAGCTGACCGTCGATAAATA CATTACCCTGCTGCCAACGCTAGCGTTGGCAGCAGGGTAATG TTTATCGACGGTCAGCTTGC (151) (152) SP-00128_Q418CGATAAAAGCCGTTGGTAGCAG CTGAACACATTACCCTGCTACC GGTAATGTGTTCAGAACGGCTTTTATCG (153) (154) SP-00114_P343 GCAAAGCGAAAGGCCAATAGCGGACCTGCGGTTCACGCTATTGG TGAACCGCAGGTC CCTTTCGCTTTGC (155) (156)SP-00112_G341 GACGATCAGCAAAGCGAAATAG CTGCGGTTCACGCGGTTGCTATCAACCGCGTGAACCGCAG TTCGCTTTGCTGATCGTC (157) (158) SP-00102_K320GCTGAATGGTAAAGAATACTAG CCTTGTTGCTCACTTTGCACTAG TGCAAAGTGAGCAACAAGGTATTCTTTACCATTCAGC (159) (160) SP-00066_F404 CTGGACAGCGACGGTAGCTAGTCAGCTTGCTATACAGAAACTAG TTCTGTATAGCAAGCTG CTACCGTCGCTGTCCAG (161) (162)SP-00113_Q342 GCAAAGCGAAAGGCTAGCCGCG CTGCGGTTCACGCGGCTAGCCT TGAACCGCAGTTCGCTTTGC  (163) (164) SP-00096_T299 GTGAGGAACAATACAATAGCTAGCACGCTCACTACGCGATACTA GTATCGCGTAGTGAGCGTGC GCTATTGTATTGTTCCTCAC (165)(166) SP-00120_Y373 GGTGAAGGGCTTTTAGCCGAGC GCGATGTCGCTCGGCTAAAAGCGACATCGC CCTTCACC (167) (168) SP-00094_N297 CGCGTGAGGAACAATACTAGAGCACTACGCGATACGTGCTCTAG CACGTATCGCGTAGTG TATTGTTCCTCACGCG (169) (170)SP-00125_F405 GACAGCGACGGTAGCTTCTAGCT GTCAGCTTGCTATACAGCTAGAGTATAGCAAGCTGAC AGCTACCGTCGCTGTC (171) (172)Extract Preparation

E. coli strain SBHS016 with modified RF1 variant A18 was harvested at afinal density of 40-55 OD, and centrifuged at 14,000 g in a SharplesModel AS14 centrifuge for 10 minutes to remove spent medium. The cellpaste was re-suspended to homogeneity with a ratio of 6 mL/g cells S30buffer (14 mM MgAcO, 60 mM KAcO, 10 mM Tris), and centrifuged again at14,000 g for 10 minutes using the Sharples AS14 for further removal ofspent medium. The resulting clarified cell paste was re-suspended in S30with a ratio of 2 mL/g cells, and the cells were lysed by single passthrough an Avestin Emulsiflex C-55 homogenizer at 17,000 psi. Thehomogenate is clarified by centrifugation at 14,000 g twice for 30minutes each, and the resulting pellets were discarded. The resultantcell extract solution was incubated at 30° C. for 2 hours, and thencentrifuged again at 14,000 g using the Sharples AS14 for particulateremoval. This final solution was frozen in LN₂ and stored at −80° C.until needed for cell-free protein synthesis.

Cell-free extracts were thawed to room temperature and incubated with 50uM iodoacetamide for 30 min. Cell-free reactions were run at 30 C for upto 10 h containing 30% (v/v) iodoacetamide-treated extract with 8 mMmagnesium glutamate, 10 mM ammonium glutamate, 130 mM potassiumglutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP,and CMP, 2 mM amino acids (1 mM for tyrosine), 4 mM sodium oxalate, 1 mMputrescine, 1.5 mM spermidine, 15 mM potassium phosphate,100 nM T7 RNAP,2.5 uM E. coli DsbC, 5 uM yeast PDI, 2 mM oxidized (GSSG) glutathioneand 15 uM yeast tRNA pN3F aminoacyl tRNA. To label synthesized proteinwith ¹⁴C, 3.33% (v/v) 1-[U-¹⁴C]-leucine (300 mCi/mmole; GE LifeSciences, Piscataway, N.J.) was added to reaction as well. Theconcentrations of heavy chain TAG variant plasmid and wild type lightchain plasmid were 7.5 ug/mL and 2.5 ug/mL respectively. As control, thecell-free expression of wild type light chain was done in parallel withTAG variants.

12 difficult to suppress sites were selected from heavy chain based onour internal study, including N297, T299, K320, G341, Q342, P343, Y373,F404, F405, S415, Q418, and V422. S136 was chosen as positive control,which has relatively high suppression. Two cell extracts, from strainsSBJY001 (which expresses wild-type RF1) and SBHS016 (which expressesmodified RF1), were used to compare the capability of the extracts toincorporate nnAA into these difficult to suppress sites.

60 uL cell-free reactions were run in 24 well plates. After thecell-free reactions were completed, TCA precipitation was performed tomeasure total and soluble proteins synthesized. In parallel, nonreducing and reducing gels were for autoradiography assay. For nonreducing gel, 4 uL of sample, 8 uL of DI H₂O and 4 uL of 4× LDS buffer(Invitrogen, Carlsbad, Calif.) were mixed before being loaded on gel.For reducing gel, 4 uL of sample, 1 uL of 1 M DTT, 7 uL of DI H₂O and 4uL of 4× LDS buffer (Invitrogen, Carlsbad, Calif.) were mixed and heatedin hot blot at 70 C for 5 minutes. Samples were analyzed by 4˜12%Bis-Tris SDS-PAGE gels (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommendations. Gels were dried and analyzed byautoradiography using a Storm 840 PhosphoImager after about 16 hoursexposure.

${{IgG}\mspace{14mu}{yield}} = \frac{\begin{matrix}{{IgG}\mspace{14mu}{full}\mspace{14mu}{length}\mspace{14mu}{band}\mspace{14mu}{intensity}} \\{{from}\mspace{14mu}{non}\mspace{14mu}{reducing}\mspace{14mu}{gel}}\end{matrix}}{\begin{matrix}{{the}\mspace{14mu}{sum}\mspace{14mu}{of}\mspace{14mu}{all}\mspace{14mu}{bands}\mspace{14mu}{intensity}\mspace{14mu}{from}} \\{{reducing}\mspace{20mu}{gel} \times \left\lbrack {{soluble}\mspace{14mu}{protein}} \right\rbrack}\end{matrix}}$

-   -   where the band intensity is determined by ImageQuant™ software        and the [soluble protein] was estimated by TCA precipitation        method.

The suppressions of amber codon at different sites of heavy chain weredetermined by [¹⁴C]-autoradiograhy of reducing SDS-AGE gels. Full lengthwild type heavy chain and suppressed heavy chain TAG variants run at 49Kd on SDS-PAGE. Non suppressed (truncated) heavy chain of TAG variantsrun at a lower molecular weight.

The  suppression  of  TAG  in  heavy  chain  is  predicted  by:${suppression} = \frac{\begin{matrix}{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{suppressed}\mspace{14mu}{heavy}} \\{{chain}\mspace{14mu}{TAG}\mspace{14mu}{variant}}\end{matrix}}{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{wild}\mspace{14mu}{type}\mspace{14mu}{heavy}\mspace{14mu}{chain}}$where the band intensity is determined by ImageQuant™ software.

To generate materials for purification, the reactions were scaled up to1 mL in 10 cm petri dishes under the same condition.

Production of tRNA

Transcriptions of all tRNA^(phe) _(CUA) transcripts were done under thefollowing conditions: 20-50 ng/μl pYD318-tRNAphe AAA-HDV-T7trm, 40 mMNaCl, 10 mM MgCl₂, 10 mM DTT, 4 mM dNTPs, 2.5 mM spermidine, 1 U/mlPPiase, 2.5 mg/ml T7 RNA polymerase, and 40 mM Tris (pH 7.9). tRNAmolecules were separated from parental RNA and HDV ribozyme RNA productby gel filtration chromatography using a tandem Sephacryl 100 or 300resin in XK50/100 columns. Sizing columns were developed in 50 mM Tris(pH 6.5) and 250 mM NaCl. Fractions containing tRNA were pooled, mixedwith 1/10 volume of 3 M sodium acetate (pH 5.2), and an equal volume ofisopropanol was added to precipitate the RNA. tRNA was stored as apellet or resuspended in 10 mM Tris (pH 6.5) and 0.1 mM EDTA.

tRNAs for use in aminoacylation reactions are treated with T4polynucleotide kinase in 100 mM MES pH 5.5, 10 mM MgCl₂ and 10 mM2-mercaptoethanol for 1 hr to remove the 2′,3′-cyclic phosphate leaving2′,3′-OH groups at the 3′ terminus of the tRNA. T4 PNK treated tRNA wasphenol:chloroform:isoamylalcohol extracted and buffer exchanged using aG25 column which removed inorganic phosphate and excess phenol. tRNA wasisopropanol or ethanol precipitated, resuspended in 10 mM Tris (pH 6.5)and 0.1 mM EDTA. The tRNA was refolded by heating the tRNA to 70° C. for20 minutes. Then 10 mM MgCl₂ was added and the mixture was slowlyequilibrated to room temperature.

HDV ribozyme cleavage of tRNA transcripts, while producing homogenous 3′ends, leaves a 2′-3′ cyclic phosphate moiety that interferes withsubsequent aminoacylation. It has been found that this can be removedusing T4 polynucleotide kinase (PNK). 40 μM tRNA was incubated at 37° C.with 0.050 mg/ml PNK in 50 mM MES (pH 5.5), 10 mM MgCl₂, 300 mM NaCl,and 0.1 mM EDTA. Dephosphorylation was assayed by two different methods.Dephosphoryation was confirmed using denaturing gel electrophoresis. Ashas been reported, dephophoylated tRNA has a reduced mobility inacid/urea gels electrophoresis. Aliquots containing 3 μg ofdephosphorylated tRNA were diluted 2-fold in loading buffer (100 mMsodium acetate (pH 5.2), 7 M urea, 1 mg/ml bromophenol blue dye) andloaded on a 6.5% 19:1 acrylamide, 100 mM sodium acetate (pH 5.2), 7 Murea gel (40 cm×34 cm) and electrophoresed overnight at 40 W. Gels werestained using 0.06% Methylene Blue, 0.5 M sodium acetate (pH 5.2) for 30minutes and destained with deionized water. Both assays indicatedsignificant dephosphorylation after only 5 minutes. Dephosphorylationwas essentially complete after 1 hour tRNA was refolded by heating to70° C., addition of 10 mM MgCl₂, and then slowly cooled to roomtemperature. RNA concentration was measured using a Nano-Drop 1000spectrophotometer (Thermo Scientific) and confirmed by gelelectrophoresis.

Amber Suppressor tRNA Aminoacylation

The conditions for non-natural aminoacylation are 50 mM HEPES pH 8.1, 40mM KCl, 75 mM MgCl₂, 5 mM ATP, 8-40 μM tRNA^(phe) _(CUA), 10 mM DTT, 2mM amino acid (pN₃F), and 40 μM PheRS T415A D243A. Determination of thepercent aminoacylation of tRNA^(phe) _(CUA) is accomplished by HPLC HICresolution of the aminoacylated and unaminoacylated moieties of tRNA.This method allows us to monitor the extent of aminoacylation of ourtRNA after is has been processed and is ready to be used forincorporation into proteins. Reactions are incubated at 37° C. for 15min and quenched with 2.5 volumes of 300 mM sodium acetate pH 5.5. Thequenched sample is extracted with 25:24:1 phenol:chloroform:isoamylalcohol pH 5.2 (ambion) and vortexed for 2 min. These are thencentrifuged at 14,000 rcf for 10-30 min at 4° C. to separate the aqueous(tRNA) and organic phases (protein). The aqueous phase (containing tRNA)is removed and added to a pre-equilibrated (300 mM NaOAC) G25 sephadexresin size exclusion column that separates based on the size of themolecule. The elutant is mixed with 2.5 volumes of 100% ethanol andincubated at −80° C. for 15-30 minutes and centrifuged at 12,000-14,000rcf for 30-45 minutes. The aminoacylated tRNA is now in a pellet thatcan be stored at −80° C. or resuspended in a slightly acidic buffer forinjection into the HPLC and/or use in OCFS reactions.

The HPLC C5 HIC column is equilibrated in buffer A (50 mM potassiumphosphate and 1.5 M ammonium sulfate pH 5.7) until the UV trace doesn'tfluctuate from zero. 1-10 μg tRNA is mixed with 100 μl of 2× buffer A(100 mM potassium phosphate and 3 M ammonium sulfate). The sample isinjected and run in a gradient from buffer A to buffer B (50 mMpotassium phosphate and 5% isopropanol) over 50 minutes.

Incorporation of pN₃F into turboGFP TAG Mutants:

To monitor fluorescence of Green Fluorescent protein in a constructwhere there is an amber codon (stop codon), the DNA encoding turboGFP(Evrogen, Russia) was cloned into our OCFS expression vector pYD317. Astop codon (TAG) was inserted by overlapping PCR mutagenesis at thenucleotides corresponding to the amino acid Lysine 37, Tyrosine 50, andGlutamate 205 (and combinations) according to the crystal structure ofturboGFP (pdb 2G6X). Therefore any suppression of the stop codon with acharged tRNA will result in fluorescence. Reactions were incubated at30° C. in a spectrophotometer (Molecular Devices, SpectraMaxM5) for fivehours with an adhesive cover (VWR, 9503130) and fluorescence intensitymeasured at 10-minute intervals, λ_(Ex)=476 nm and λ_(Em)=510. OCFSreaction mix was immediately added to microplate with inhibitor for a 25μL final reaction volume containing 30% S30 extract, 24 ug/mL T7 RNApolymerase, 1 mM L-tyrosine (Sigma, T8566), pre-mix*, 10-60 μMpN₃F-tRNA^(phe) _(CUA) or uncharged tRNA^(phe) _(CUA), and 3 nM turboGFPplasmid in DEPC-treated water (G Biosciences, 786-109). A positivecontrol reaction using turboGFP without the stop codon was used toensure that the reactions proceeded with rates similar previouslyobserved, while reactions containing turboGFP Y50TAG were also runwithout tRNA to ensure no fluorescence was detected (negative control).Suppression efficiencies were calculated by comparison of positivecontrol fluorescence to the amber codon containing templatefluorescence.

As shown in FIG. 2, expression of the IgG heavy chain TAG variants inthe SBJY001 cell free extract, which expresses OmpT1 and wild-type RF-1,resulted in relatively poor yield of soluble full length IgG. Incontrast, expression of the IgG heavy chain TAG variants in the SBHS016cell free extract, which expresses OmpT1 and a modified RF-1 protein(variant A18) containing the triple substitution N296K/L297R/L298R,resulted in relatively high yield of full length IgG. Further, as shownin FIG. 3, expression of the IgG heavy chain TAG variants in the SBJY001cell free extract resulted in relatively poor amber suppression (i.e.,the heavy chain protein was truncated). In contrast, expression of theIgG heavy chain TAG variants in the SBHS016 cell free extract resultedin relatively high amber suppression (i.e., much less of the heavy chainprotein was truncated), which corresponds to the substantially higheryields of full length IgG observed in FIG. 2.

This example demonstrates that OmpT1 cleavage of RF1 provides a dramaticimprovement in the yield of heavy chains incorporating the desired nnAAat an amber codon in the coding sequence, as compared to intact RF1 thatis not cleavable by OmpT1.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

LIST OF SEQUENCES

Escherichia coli Release Factor 1 (RF1) SEQ ID NO: 1MKPSIVAKLEALHERHEEVQALLGDAQTIADQERFRALSREYAQLSDVSRCFTDWQQVQEDIETAQMMLDDPEMREMAQDELREAKEKSEQLEQQLQVLLLPKDPDDERNAFLEVRAGTGGDEAALFAGDLFRMYSRYAEARRWRVEIMSASEGEHGGYKEIIAKISGDGVYGRLKFESGGHRVQRVPATESQGRIHTSACTVAVMPELPDAELPDINPADLRIDTFRSSGAGGQHVNTTDSAIRITHLPTGIVVECQDERSQHKNKAKALSVLGARIHAAEMAKRQQAEASTRRNLLGSGDRSDRNRTYNFPQGRVTDHRINLTLYRLDEVMEGKLDMLIEPIIQEHQADQLAALSEQE Escherichia coli Release Factor 2 (RF2)SEQ ID NO: 2 MFEINPVNNRIQDLTERSDVLRGYLDYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLEAVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADCYLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESEGEVAGIKSVTIKISGDYAYGWLRTETGVHRLVRKSPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRTSGAGGQHVNRTESAVRITHIPTGIVTQCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWGSQIRSYVLDDSRIKDLRTGVETRNTQAVLDGSLDQFIEASLKAGLEscherichia coli Outer Membrane Protein T1 (OmpT)(signal peptide underlined) SEQ ID NO: 3MRAKLLGIVLTTPIAISSFASTETLSFTPDNINADISLGTLSGKTKERVYLAEEGGRKVSQLDWKFNNAAIIKGAINWDLMPQISIGAAGWTTLGSRGGNMVDQDWMDSSNPGTWTDESRHPDTQLNYANEFDLNIKGWLLNEPNYRLGLMAGYQESRYSFTARGGSYIYSSEEGFRDDIGSFPNGERAIGYKQRFKMPYIGLTGSYRYEDFELGGTFKYSGWVESSDNDEHYDPGKRITYRSKVKDQNYYSVAVNAGYYVTPNAKVYVEGAWNRVTNKKGNTSLYDHNNNTSDYSKNGAGIENYNFITTAGLKYTFSwitch loop region of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 4 QQAEASTRRNLLGSGDRSN296K mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 5 QQAEASTRRKLLGSGDRSN296R mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 6 QQAEASTRRRLLGSGDRSL297K mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 7 QQAEASTRRNKLGSGDRSL297R mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 8 QQAEASTRRNRLGSGDRSL297V mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 9 QQAEASTRRNVLGSGDRSL298K mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 10 QQAEASTRRNLKGSGDRSL298R mutation in the switch loop region of ReleaseFactor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 11 QQAEASTRRNLRGSGDRSN296K, L297K mutation in the switch loop region ofRelease Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 12QQAEASTRRKKLGSGDRS N296K, L297R = A13 mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 13QQAEASTRRKRLGSGDRS N296K, L297V mutation in the switch loop region ofRelease Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 14QQAEASTRRKVLGSGDRS N296K, L297V mutation in the switch loop region ofRelease Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 15QQAEASTRRRKLGSGDRS N296R, L297R mutation in the switch loop region ofRelease Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 16QQAEASTRRRRLGSGDRS N296R, L297V mutation in the switch loop region ofRelease Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 17QQAEASTRRRVLGSGDRS N296K, L297K, L298K mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 18QQAEASTRRKKKGSGDRS N296K, L297K, L298R mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 19QQAEASTRRKKRGSGDRS N296K, L297R, L298K = A17 mutation in the switchloop region of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 20 QQAEASTRRKRKGSGDRS N296K, L297R, L298R =A18 mutation in the switch loop region of Release Factor 1 (RF1), aminoacids 287 to 304 SEQ ID NO: 21 QQAEASTRRKRRGSGDRSN296R, L297R, L298R mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 22QQAEASTRRRRRGSGDRS N296R, L297K, L298R mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 23QQAEASTRRRKRGSGDRS N296R, L297R, L298K mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 24QQAEASTRRRRKGSGDRS N296R, L297K, L298K mutation in the switch loopregion of Release Factor 1 (RF1), amino acids 287 to 304 SEQ ID NO: 25QQAEASTRRRKKGSGDRS OmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 26 QARRGSTRRNLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 27 QQARRGTRRNLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 28 QQAARRGRRNLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 29 QQAEARRGRNLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 30 QQAEAARRGNLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 31 QQAEASARRGLLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 32 QQAEASTARRGLGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 33 QQAEASTRARRGGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 34 QQAEASTRRARRGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 35 QQAEASTRRNARRGGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 36 QQAEASTRRNLARRGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 37 QQAEASTRRNLLARRGRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 38 QQAEASTRRNLLGARRGSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 39 QQAEASTRRNLLGSARRGOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 40 QQAEASTRRNLLGSGARROmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 41 QQAWLAARRGRGGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 42 QQAEWLAARRGRGSGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 43 QQAEAWLAARRGRGGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 44 QQWGGRWARKKGTIGDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 45 QQAWGGRWARKKGTIDRSOmpT cleavage peptide sequence inserted in theswitch loop of Release Factor 1 (RF1), amino acids 287 to 304SEQ ID NO: 46 QQAEWGGRWARKKGTIRS OmpT cleavage peptide SEQ ID NO: 47ARRG OmpT cleavage peptide SEQ ID NO: 48 WLAARRGRG OmpT cleavage peptideSEQ ID NO: 49 WGGRWARKKGTI C-terminal sequence of short form of λphage Gam protein (GamS) SEQ ID NO: 50 GGSHHHHHH short form of λphage Gam protein (GamS) gene amplification primer SEQ ID NO: 51ATATATCATATGAACGCTTATTACATTCAGGATCGTCTTGAG short form of λphage Gam protein (GamS) gene amplification primer SEQ ID NO: 52ATATATGTCGACTTAATGATGATGATGATGATGAGAACCCCCTACCTCTGAATCAATATCAACCTGGTGGTG 5′-fragment including T7 promoter, constant region ofthe N-terminal sequence and the mutation site firststep PCR amplification primer 5chiT2PT7 SEQ ID NO: 53GCGTACTAGCGTACCACGTGGCTGGTGGCCGATTCATTAATGCAGCTGGCACGAC AGG3′-fragment including mutation site, constant C-terminalregion and T7 terminator sequences first step PCRamplification primer 3chiT2TT7 SEQ ID NO: 54GCGTACTAGCGTACCACGTGGCTGGTGGCGGTGAGTTTTCTCCTTCATTACAGAA ACGGCsingle primer 5chiT2 for 5′-fragment and 3′-fragmentassembly by overlapping PCR SEQ ID NO: 55 GCGTACTAGCGTACCACGTGGCTGGTGGWT RF1 variant OmpT cleavage site screening forward primer SEQ ID NO: 56GCTCGATGATCCTGAAATGCGTGAGATGGCGCAGGM74R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 57 GCTCGATGATCCTGAACGCCGTGAGATGGCGCAGGE76K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 58 CGATGATCCTGAAATGCGTAAGATGGCGCAGGATGAACE84K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 59 CAGGATGAACTGCGCAAAGCTAAAGAAAAAAGCGAGCAACA85R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 60 CAGGATGAACTGCGCGAACGTAAAGAAAAAAGCGAGCAACE87R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 61 GGATGAACTGCGCGAAGCTAAACGTAAAAGCGAGCAACTGGAACE108R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 62 GCCAAAAGATCCTGATGACCGTCGTAACGCCTTCCTCGT293R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 63 CAACAGGCCGAAGCGTCTCGCCGTCGTAACCTGCN296K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 64 GCGTCTACCCGTCGTAAACTGCTGGGGAGTGGCGS304K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 65 GGGAGTGGCGATCGCAAGGACCGTAACCGTACTTACN296K/L297V RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 66 CGAAGCGTCTACCCGTCGTAAAGTTCTGGGGAGTGGCGATCGCAGCN296K/L297K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 67 CGAAGCGTCTACCCGTCGTAAAAAGCTGGGGAGTGGCGATCGCAGCN296K/L297R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 68 CGAAGCGTCTACCCGTCGTAAACGTCTGGGGAGTGGCGATCGCAGCN296R/L297V RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 69 CGAAGCGTCTACCCGTCGTCGCGTTCTGGGGAGTGGCGATCGCAGCN296R/L297K RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 70 CGAAGCGTCTACCCGTCGTCGCAAGCTGGGGAGTGGCGATCGCAGCN296R/L297R RF1 variant OmpT cleavage site screening forward primerSEQ ID NO: 71 CGAAGCGTCTACCCGTCGTCGCCGTCTGGGGAGTGGCGATCGCAGCN296K/L297R/L298K RF1 variant OmpT cleavage sitescreening forward primer SEQ ID NO: 72CAGGCCGAAGCGTCTACCCGTCGTAAACGTAAGGGGAGTGGCGATCGCAGCGAC CN296K/L297R/L298R RF1 variant OmpT cleavage sitescreening forward primer SEQ ID NO: 73CAGGCCGAAGCGTCTACCCGTCGTAAACGTCGCGGGAGTGGCGATCGCAGCGAC CWT RF1 variant OmpT cleavage site screening reverse primer SEQ ID NO: 74CCTGCGCCATCTCACGCATTTCAGGATCATCGAGCM74R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 75 CCTGCGCCATCTCACGGCGTTCAGGATCATCGAGCE76K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 76 GTTCATCCTGCGCCATCTTACGCATTTCAGGATCATCGE84K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 77 GTTGCTCGCTTTTTTCTTTAGCTTTGCGCAGTTCATCCTGA85R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 78 GTTGCTCGCTTTTTTCTTTACGTTCGCGCAGTTCATCCTGE87R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 79 GTTCCAGTTGCTCGCTTTTACGTTTAGCTTCGCGCAGTTCATCCE108R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 80 CGAGGAAGGCGTTACGACGGTCATCAGGATCTTTTGGCT293R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 81 GCAGGTTACGACGGCGAGACGCTTCGGCCTGTTGN296K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 82 CGCCACTCCCCAGCAGTTTACGACGGGTAGACGCS304K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 83 GTAAGTACGGTTACGGTCCTTGCGATCGCCACTCCCN296K/L297V RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 84 GCTGCGATCGCCACTCCCCAGAACTTTACGACGGGTAGACGCTTCGN296K/L297K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 85 GCTGCGATCGCCACTCCCCAGCTTTTTACGACGGGTAGACGCTTCGN296K/L297R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 86 GCTGCGATCGCCACTCCCCAGACGTTTACGACGGGTAGACGCTTCGN296R/L297V RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 87 GCTGCGATCGCCACTCCCCAGAACGCGACGACGGGTAGACGCTTCGN296R/L297K RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 88 GCTGCGATCGCCACTCCCCAGCTTGCGACGACGGGTAGACGCTTCGN296R/L297R RF1 variant OmpT cleavage site screening reverse primerSEQ ID NO: 89 GCTGCGATCGCCACTCCCCAGACGGCGACGACGGGTAGACGCTTCGN296K/L297R/L298K RF1 variant OmpT cleavage sitescreening reverse primer SEQ ID NO: 90GGTCGCTGCGATCGCCACTCCCCTTACGTTTACGACGGGTAGACGCTTCGGCCTGN296K/L297R/L298R RF1 variant OmpT cleavage sitescreening reverse primer SEQ ID NO: 91GGTCGCTGCGATCGCCACTCCCGCGACGTTTACGACGGGTAGACGCTTCGGCCTGNo. 0 RF1 variant OmpT cleavage peptide insertion forward oligo primerSEQ ID NO: 92 GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 1 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 93CGCCGTGGTTCTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 2 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 94GCACGCCGTGGTACCCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 3 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 95GCCGCACGCCGTGGTCGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 4 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 96GCCGAAGCACGCCGTGGTCGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 5 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 97GCCGAAGCGGCACGCCGTGGTAACCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 6 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 98GCCGAAGCGTCTGCACGCCGTGGTCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 7 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 99GCCGAAGCGTCTACCGCACGCCGTGGTCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 8 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 100GCCGAAGCGTCTACCCGTGCACGCCGTGGTGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 9 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 101GCCGAAGCGTCTACCCGTCGTGCACGCCGTGGTAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 10 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 102GCCGAAGCGTCTACCCGTCGTAACGCACGCCGTGGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 11 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 103GCCGAAGCGTCTACCCGTCGTAACCTGGCACGCCGTGGTGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 12 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 104GCCGAAGCGTCTACCCGTCGTAACCTGCTGGCACGCCGTGGTCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 13 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 105GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGGCACGCCGTGGTAGCGACCGTAACCGTACTTACAACTTCCCG No. 14 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 106GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGCACGCCGTGGTGACCGTAACCGTACTTACAACTTCCCG No. 15 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 107GCCGAAGCGTCTACCCGTCGTAACCTGCTGGGGAGTGGCGCACGCCGTGACCGTAACCGTACTTACAACTTCCCG No. 16 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 108GCCGAAGCGTCTACCCGTCGTCGTCTGCTGGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 17 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 109GCCTGGCTGGCAGCGCGTCGCGGTCGTGGCGGGAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 18 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 110GCCGAATGGCTGGCAGCGCGTCGCGGTCGTGGCAGTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 19 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 111GCCGAAGCGTGGCTGGCAGCGCGTCGCGGTCGTGGCGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 20 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 112TGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTGGCGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 21 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 113GCCTGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTGATCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 22 RF1 variant OmpT cleavage peptide insertionforward oligo primer SEQ ID NO: 114GCCGAATGGGGTGGCCGTTGGGCTCGCAAGAAAGGTACTATTCGCAGCGACCGTAACCGTACTTACAACTTCCCG No. 0 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 115GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 1 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 116GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTAGAACCACGGCGTGCTTGGCGTTTTGCCATTTCAGCAGC No. 2 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 117GCTGCGATCGCCACTCCCCAGCAGGTTACGACGGGTACCACGGCGTGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 3 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 118GCTGCGATCGCCACTCCCCAGCAGGTTACGACGACCACGGCGTGCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 4 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 119GCTGCGATCGCCACTCCCCAGCAGGTTACGACCACGGCGTGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 5 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 120GCTGCGATCGCCACTCCCCAGCAGGTTACCACGGCGTGCCGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 6 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 121GCTGCGATCGCCACTCCCCAGCAGACCACGGCGTGCAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 7 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 122GCTGCGATCGCCACTCCCCAGACCACGGCGTGCGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 8 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 123GCTGCGATCGCCACTCCCACCACGGCGTGCACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 9 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 124GCTGCGATCGCCACTACCACGGCGTGCACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 10 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 125GCTGCGATCGCCACCACGGCGTGCGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 11 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 126GCTGCGATCACCACGGCGTGCCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 12 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 127GCTGCGACCACGGCGTGCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 13 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 128GCTACCACGGCGTGCCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 14 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 129ACCACGGCGTGCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 15 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 130ACGGCGTGCGCCACTCCCCAGCAGGTTACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 16 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 131GCTGCGATCGCCACTCCCCAGCAGACGACGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 17 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 132GCTGCGATCGCCACTCCCGCCACGACCGCGACGCGCTGCCAGCCAGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 18 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 133GCTGCGATCGCCACTGCCACGACCGCGACGCGCTGCCAGCCATTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 19 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 134GCTGCGATCGCCGCCACGACCGCGACGCGCTGCCAGCCACGCTTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 20 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 135GCTGCGATCGCCAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCACTGTTGGCGTTTTGCCATTTCAGCAGC No. 21 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 136GCTGCGATCAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCAGGCCTGTTGGCGTTTTGCCATTTCAGCAGC No. 22 RF1 variant OmpT cleavage peptide insertionreverse oligo primer SEQ ID NO: 137GCTGCGAATAGTACCTTTCTTGCGAGCCCAACGGCCACCCCATTCGGCCTGTTGGCGTTTTGCCATTTCAGCAGC RF1 amplification primer 5His-RF1 SEQ ID NO: 138CATATGCATCACCATCACCATCACGGTGGTGGCTCTAAGCCTTCTATCGTTGCCA AACTGGAAGCCRF1 amplification primer 3RF1 SEQ ID NO: 139GTCGACTTATTCCTGCTCGGACAACGCCGCCAGOligonucleotide-Mediated Allelic Replacement (OMAR) PCR oligo 1opRF1 KRSEQ ID NO: 140GGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCaAGacGcTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCOligonucleotide-Mediated Allelic Replacement (OMAR) PCR oligo 1opRF1 KRRSEQ ID NO: 141GGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCacgacGcTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCOligonucleotide-Mediated Allelic Replacement (OMAR) PCR oligo 1opRF1 KRKSEQ ID NO: 142GGGAAGTTGTAAGTACGGTTACGGTCGCTGCGATCcCCtgaaCCcttacGcTTtCGACGGGTAGACGCTTCGGCCTGTTGGCGTTTTGCCMismatch Amplification Mutation Assay (MAMA) PCR oligo 3KR op-PCRSEQ ID NO: 143 GCG ATC CCC TGA ACC AAG ACG CMismatch Amplification Mutation Assay (MAMA) PCR oligo 3 KRR op-PCRSEQ ID NO: 144 CGATCcCCtgaaCCacgacGcMismatch Amplification Mutation Assay (MAMA) PCR oligo 3KRK op-PCRSEQ ID NO: 145 TGCGATCcCCtgaaCCcttacGcMismatch Amplification Mutation Assay (MAMA) PCR oligo 5 RF1 op-PCRSEQ ID NO: 146 CGTGACGGGGATAACGAACGCCrolling circle amplification and primer extension sequencing primer T7SEQ ID NO: 147 TAATACGACTCACTATAGGrolling circle amplification and primer extensionsequencing primer T7 term SEQ ID NO: 148 GCTAGTTATTGCTCAGCGSite directed mutagenesis variant SP-00067_V422 sense oligoSEQ ID NO: 149 CGTTGGCAGCAGGGTAATTAGTTCAGCTGCAGCGTTATGSite directed mutagenesis variant SP-00067 V422 antisense oligoSEQ ID NO: 150 CATAACGCTGCAGCTGAACTAATTACCCTGCTGCCAACGSite directed mutagenesis variant SP-00127_S415 sense oligoSEQ ID NO: 151 GCAAGCTGACCGTCGATAAATAGCGTTGGCAGCAGGGTAATGSite directed mutagenesis variant SP-00127_S415 antisense oligoSEQ ID NO: 152 CATTACCCTGCTGCCAACGCTATTTATCGACGGTCAGCTTGCSite directed mutagenesis variant SP-00128_Q418 sense oligoSEQ ID NO: 153 CGATAAAAGCCGTTGGTAGCAGGGTAATGTGTTCAGSite directed mutagenesis variant SP-00128_Q418 antisense oligoSEQ ID NO: 154 CTGAACACATTACCCTGCTACCAACGGCTTTTATCGSite directed mutagenesis variant SP-00114_P343 sense oligoSEQ ID NO: 155 GCAAAGCGAAAGGCCAATAGCGTGAACCGCAGGTCSite directed mutagenesis variant SP-00114_P343 antisense oligoSEQ ID NO: 156 GACCTGCGGTTCACGCTATTGGCCTTTCGCTTTGCSite directed mutagenesis variant SP-00112_G341 sense oligoSEQ ID NO: 157 GACGATCAGCAAAGCGAAATAGCAACCGCGTGAACCGCAGSite directed mutagenesis variant SP-00112_G341 antisense oligoSEQ ID NO: 158 CTGCGGTTCACGCGGTTGCTATTTCGCTTTGCTGATCGTCSite directed mutagenesis variant SP-00102_K320 sense oligoSEQ ID NO: 159 GCTGAATGGTAAAGAATACTAGTGCAAAGTGAGCAACAAGGSite directed mutagenesis variant SP-00102_K320 antisense oligoSEQ ID NO: 160 CCTTGTTGCTCACTTTGCACTAGTATTCTTTACCATTCAGCSite directed mutagenesis variant SP-00066_F404 sense oligoSEQ ID NO: 161 CTGGACAGCGACGGTAGCTAGTTTCTGTATAGCAAGCTGSite directed mutagenesis variant SP-00066_F404 antisense oligoSEQ ID NO: 162 CAGCTTGCTATACAGAAACTAGCTACCGTCGCTGTCCAGSite directed mutagenesis variant SP-00113_Q342 sense oligoSEQ ID NO: 163 GCAAAGCGAAAGGCTAGCCGCGTGAACCGCAGSite directed mutagenesis variant SP-00113_Q342 antisense oligoSEQ ID NO: 164 CTGCGGTTCACGCGGCTAGCCTTTCGCTTTGCSite directed mutagenesis variant SP-00096_T299 sense oligoSEQ ID NO: 165 GTGAGGAACAATACAATAGCTAGTATCGCGTAGTGAGCGTGCSite directed mutagenesis variant SP-00096_T299 antisense oligoSEQ ID NO: 166 GCACGCTCACTACGCGATACTAGCTATTGTATTGTTCCTCACSite directed mutagenesis variant SP-00120_Y373 sense oligoSEQ ID NO: 167 GGTGAAGGGCTTTTAGCCGAGCGACATCGCSite directed mutagenesis variant SP-00120_Y373 antisense oligoSEQ ID NO: 168 GCGATGTCGCTCGGCTAAAAGCCCTTCACCSite directed mutagenesis variant SP-00094_N297 sense oligoSEQ ID NO: 169 CGCGTGAGGAACAATACTAGAGCACGTATCGCGTAGTGSite directed mutagenesis variant SP-00094_N297 antisense oligoSEQ ID NO: 170 CACTACGCGATACGTGCTCTAGTATTGTTCCTCACGCGSite directed mutagenesis variant SP-00125_F405 sense oligoSEQ ID NO: 171 GACAGCGACGGTAGCTTCTAGCTGTATAGCAAGCTGACSite directed mutagenesis variant SP-00125_F405 antisense oligoSEQ ID NO: 172 GTCAGCTTGCTATACAGCTAGAAGCTACCGTCGCTGTC

What is claimed is:
 1. A mutant Releasing Factor 1 (RF1) protein,wherein the mutant RF1 protein comprises an amino acid sequence havingat least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, wherein the mutant RF1 protein has activity to recognize a stop codonin an mRNA sequence and terminate translation, wherein the amino acidsequence of the mutant RF1 protein corresponding to amino acids 287-304of SEQ ID NO: 1 comprises a cleavage site for a wild-type Outer MembraneProtein T1 (OmpT1), wherein the amino acid sequence of the cleavage sitefor the wild-type OmpT1 comprises an amino acid sequence selected fromthe group consisting of ARR, ARRG (SEQ ID NO:47), WLAARRGRG (SEQ IDNO:48), and WGGRWARKKGTI (SEQ ID NO:49).
 2. The mutant RF1 protein ofclaim 1, wherein the amino acid sequence of the cleavage site for thewild-type OmpT1 comprises an amino acid sequence selected from the groupconsisting of: QARRGSTRRNLLGSGDRS (SEQ ID NO:26); QQARRGTRRNLLGSGDRS(SEQ ID NO:27); QQAARRGRRNLLGSGDRS (SEQ ID NO:28); QQAEARRGRNLLGSGDRS(SEQ ID NO:29); QQAEAARRGNLLGSGDRS (SEQ ID NO:30); QQAEASARRGLLGSGDRS(SEQ ID NO:31); QQAEASTARRGLGSGDRS (SEQ ID NO:32); QQAEASTRRARRGSGDRS(SEQ ID NO:34); QQAEASTRRNARRGGDRS (SEQ ID NO:35); QQAEASTRRNLARRGDRS(SEQ ID NO:36); QQAEASTRRNLLARRGRS (SEQ ID NO:37); QQAEASTRRNLLGARRGS(SEQ ID NO:38); QQAEASTRRNLLGSARRG (SEQ ID NO:39); QQAEASTRRNLLGSGARR(SEQ ID NO:40) QQAWLAARRGRGGSGDRS (SEQ ID NO:41); QQAEWLAARRGRGSGDRS(SEQ ID NO:42); QQAEAWLAARRGRGGDRS (SEQ ID NO:43) QQWGGRWARKKGTIGDRS(SEQ ID NO:44); QQAWGGRWARKKGTIDRS (SEQ ID NO:45); andQQAEWGGRWARKKGTIRS (SEQ ID NO:46).
 3. A bacterial cell comprising bothan OmpT1 and the mutant RF1 protein of claim
 1. 4. A nucleic acidencoding the mutant RF1 protein of claim
 1. 5. A bacterial cellcomprising the nucleic acid of claim 4, wherein the nucleic acid isincorporated into a genome of the bacterial cell.
 6. A bacterial cellextract comprising the mutant RF1 protein of claim
 1. 7. A cell freesynthesis system comprising in a single reaction mixture: i) componentsfrom a bacterial lysate sufficient to translate a nucleic acid templateencoding a protein; ii) a nucleic acid template encoding a protein ofinterest and having at least one amber codon; iii) tRNA complementary tothe amber codon; and, iv) the mutant RF1 protein of claim
 1. 8. The cellfree synthesis system of claim 7, wherein the single reaction mixturefurther comprises a non-natural amino acid and corresponding amino acidtRNA synthetase, the tRNA synthetase able to charge the tRNAcomplimentary to the amber codon with the non-natural amino acid.
 9. Thecell free synthesis system of claim 8, wherein the system generates ATPvia an active oxidative phosphorylation system.
 10. The mutant RF1protein of claim 1, wherein the wild-type OmpT1 comprises the amino acidsequence of SEQ ID NO:
 3. 11. A method for expressing a protein ofinterest in a bacterial cell-free synthesis system, comprising: i.combining a nucleic acid template encoding the protein of interest witha bacterial cell free synthesis extract to produce a bacterial cell-freesynthesis system, wherein the bacterial cell free synthesis extractcomprises the mutant RF1 protein of claim 1 and a wild-type OmpT1; ii.allowing the mutant RF1 protein to be cleaved by the wild-type OmpT1;and iii. expressing the protein of interest from the nucleic acidtemplate.
 12. A method for preparing a cell free synthesis extract, themethod comprising the steps of: i) culturing OmpT1 positive bacteriacomprising the nucleic acid of claim 4 to express the mutant RF1protein; and ii) lysing the OmpT1 positive bacteria to create the cellfree synthesis extract.
 13. The method of claim 12, wherein the OmpT1positive bacteria is Escherichia coli.